Method for treatment of aneurysms

ABSTRACT

The present invention generally concerns the detection and/or treatment of aneurysm in a non-invasive manner. In particular cases, the invention concerns methods and compositions for localizing a labeled composition to the site of an aneurysm for its detection and, in further cases, treatment of the aneurysm. In specific cases, the composition targets a subendothelial component of the aneurysmal wall, such as a smooth muscle cell exposed at the luminal surface of the vessel. In further specific cases, the composition targets an integrin receptor or laminin.

The present application is a divisional application of U.S. patentapplication Ser. No. 12/809,139, filed Jun. 18, 2010, which is anational phase application filed under 35 USC §371 fromPCT/US2008/084445, filed Nov. 23, 2008, which claims priority to U.S.Provisional Patent Application Ser. No. 61/016,090, filed Dec. 21, 2007,all of which applications are incorporated by reference herein in theirentirety.

TECHNICAL FIELD

The present invention generally concerns at least the fields ofmedicine, cell biology, and molecular biology. In particular aspects,the present invention concerns the detection and/or treatment ofcerebral aneurysms.

BACKGROUND OF THE INVENTION

Annually in the U.S., aneurysmal subarachnoid hemorrhage affects greaterthan 30,000 people. Ten to 15 percent of these individuals die beforereaching the hospital and greater than 50 percent die within the firstmonth following rupture. Of those patients that survive, approximatelyhalf suffer some permanent neurological deficit.

Intra-cranial saccular (berry) aneurysm is a balloon-like distension ofa major brain artery occurring at (or near) the apex of arterial forks.It is frequently (90%) located on the anterior part of the circle ofWillis. Various hypothesis have been proposed regarding thedevelopmental mechanisms of Saccular Cerebral Artery Aneurysms (SCAAs)such as the medial defect theory (Forbus, 1930), the elastic lamellartheory (Glynn, 1940), degenerative theory (Stehbens, 1963; Stehbens,1972), congenital theories (Bremer, 1943; Agnoli, 1982), and others(Sekha et al., 1981). Recently the development of an experimental animalmodel of the disease with pathological features very similar to those ofhuman SCCA has made possible the study of the pathogenesis of humanSCCAs (Hashimoto et al., 1978). It has been shown that hemodynamicstress induces the development of cerebral aneurysms causingdegenerative changes of the endothelium, the elastic lamina and themedial smooth muscle cells at specific site on the arterial bifurcation(Kojima et al., 1988).

Histological Features of SCAAs

The anterior cerebral artery/olfactory artery (ACA/OA) junction is oneof the most common sites of aneurysm development in the animal model.Its normal structure and changes due to aneurysm development have beenwidely studied.

1. Normal ACA/OA Artery Junction

The apex of a normal ACA/OA junction consists of normal arterialcomponents (endothelial cells, internal elastic lamina, medial smoothmuscle cells, and thin adventitial fibrous connective tissue). In theapical region, there is an intimal protrusion called pad consistentlylocated near the apex on the distal side of the ACA. This pad iscomposed of spindle-shaped cells similar to the medial smooth musclecells, rich in interstitial tissue. Under and just distal to the intimalpad on the side of the ACA, the internal elastic lamina is thinned andfragmented.

2. Aneurysm Formation at ACA/OA Artery Junction

The initial changes of aneurysm occur at the intimal pad and theneighboring distal portion. In the histological structure of early stageaneurysms, there are the following characteristics: 1) the wall does notsignificantly protrude; 2) initial changes are localized almostexclusively at the intimal pad and its neighboring distal portion; and3) there is fragmentation of internal elastic lamina and slight thinningof the media (decrease of medial smooth muscle cells in number). In thehistological structure of advanced stage aneurysms, there are thefollowing characteristics: 1) aneurismal wall consists only of a fibrousadventitia and a layer of endothelial cells; 2) complete disappearanceof the Internal Elastic Lamina (I.E.L) at the level of the aneurismalneck; and 3) media layer (smooth muscle cells (SMC)) ceases abruptlyproximal to the neck.

The histological features of SCAAs include degenerative changes ofendothelium, fragmentation and disappearance of I.E.L, and thinning(then disappearance) of medial layer. In degenerative changes of theendothelium, the following has been observed. Severe changes inendothelium have been reported. Nagata et al. (1981) examined byscanning electron microscopy the luminal surface of the cerebralaneurysms. They noticed some variations in the shape of the endothelialcells from fusiform to polygonal. Some of them showed balloon-likeprotrusions. Crater-like depressions on the endothelial surface andsmall holes and enlarged gaps at the junction of the endothelial cellswere frequently observed. Gap formation at the junctions between theendothelial cells was one of the most obvious changes on the luminalsurface of the aneurysms. Kojima et al. (1986) studying various stage ofearly aneurismal changes reported alterations of the endotheliumdeveloping just distal to intimal pad. Degenerated cells with balloonsand craters were observed intermingled with regenerated endothelialcells. Interendothelial gaps were also seen. They concluded that somehemodynamic stress, possibly turbulent flow or secondary flow, mayinjure the endothelial cells located distal to the pad, and such injuredendothelial cells in turn develop saccular cerebral aneurysms. Greenhilland Stehbens (1982) also described severe alterations of the endotheliumand subendothelial tissues caused by hemodynamic stress. Kim et al.(1992) studied aneurismal changes in experimental monkeys and foundendothelial injury. They suggested that aneurismal changes are initiatedby degenerative changes in the endothelium, which are followed byalterations in the underlying elastic lamina and, in turn, in the mediallayer.

Degenerative changes of the internal elastic lamina and the medialsmooth muscle cells are also known. Hazama et al. (1986) showed thatearly aneurismal changes consist on degenerative changes of the InternalElastic Lamina (I.E.L) at the intimal pad and the neighboring areadistal to the pad associated to regressive changes of medial smoothmuscle layer. Kim et al. (1988) also reported degenerative changes ofthe I.E.L and medial smooth muscle layer. Morimoto et al. (2002) foundthat the characteristic of SCAA formation in a mouse model was thinningof medial smooth muscle layer and disappearance of the I.E.L. Kondo etal. (1998) found that the histological features of aneurismal changeswere thinning of the medial layer accompanied by fragmentation ordisappearance of internal elastic lamina with wall dilatation. Theynoted a decreased number of SMCs in the medial layer due to apoptosis.They concluded that the death of medial SMCs through apoptosis plays animportant role in aneurysm formation.

Molecular Mechanisms of SCAAs Formation

While the pathological features of aneurismal lesions described aboveare well documented, the precise molecular mechanisms involved in theformation of cerebral aneurysms have not yet been conclusivelyidentified. Hemodynamic stress has been shown in many investigations tobe the major cause of various degenerative changes in SCAA formation(Nakatani et al., 1991; Stehbens, 1989). This hemodynamic stress mightinduce a complex, multifactorial remodeling through a variety ofmediators and pathways. Recent studies have reported the role of nitricoxide in the development of SCAA. Inducible NO synthase (iNOS) wasinduced in response to hemodynamic stress and NO synthesized by iNOSserves to damage the arterial wall and lead to aneurysm formation(Fukuda et al., 2000). Other molecular mechanisms such as activeexpressions of matrix metalloproteinases (Houghton et al., 2006),apoptosis of medial smooth muscle cells (Cohen et al., 1991) have beenshown associated with SCAA. The role of elastase in the degradation ofI.E.L in early aneurismal lesions has also been discussed. Nagata et al.(1981) reported that in experimental aneurysms many leukocytes werepresent adhering to the inter endothelial gaps, which may represent theparticipation of leukocytes in degradation of the I.E.L. Cajander andHassler (1976) also found extracellular lysosome-like granules closelyconnected to the disintegrated elastic lamella in the mouths ofaneurysms and hypothesized that discharged leukocyte granules containingelastase help to destroy the elastic lamella. Enhanced activity ofelastase in the arterial wall may also participate in the degenerativechanges of the internal elastic lamina, as in the case of hypertension(Yamada et al., 1983).

Two studies have brought significant insights into the mechanism offormation of cerebral aneurysms.

1. Futami et al. (1995) have demonstrated that fibronectin (as well ascollagen IV and I) normally expressed in the subendothelium of artery,disappears in early aneurysmal lesions. The absence of fibronectin inthe aneurysm wall is a critical feature in aneurysm formationconsidering the role of this Extra-cellular Matrix (ECM) protein inwound repair and its role in modulation of SMC phenotype (see below).

2. Jamous et al. (2007) have demonstrated the sequence ofultrastructural, morphological and pathological changes leading to theformation of saccular intracranial aneurysms in vivo. They used thecurrent established animal model to induce cerebral aneurysm. Theystudied the anterior cerebral artery-olfactory artery bifurcationmorphologically by using vascular corrosion casts andimmunohistochemically by using specific antibodies against endothelialnitric oxide synthase (eNOS), α-smooth muscle actin (α-SMA: marker ofSMCs), macrophages, and matrix metalloproteinase-9. They showed that theformation of intracranial aneurysms starts with endothelial injury atthe apical intimal pad (evidenced by the loss of eNOS expression) (stageI); this leads to the formation of an inflammatory zone. Thisinflammatory zone shows subendothelial expression of α-SMA and a loss ofeNOS. There is no protusion on the vessel wall at this earlyinflammatory stage (stage IIA). The progression of inflammation resultsin arterial wall destruction and the development of a defect presentingas a narrow slit; this is associated with protusion of the vessel wall(Stage IIB). This defect is continuous with the lumen of the parentartery, lacks eNOS expression, and contained α-SMA positive SMCs andmacrophages. Expansion of this defect results in the formation of asaccular dilatation (stage III). The walls of the cavity continued tolack eNOS expression, contained a layer of α-SMA-positive SMCs and arepositive for MMP-9 expression. The authors suggested that endothelialinjury and exposure of the subendothelial matrix initiate plateletactivation and adhesion. Activated platelets secrete growth factors thatcontribute to the recruitment of macrophages and promote migration ofSMCs. These processes result in the formation of the inflammatory zone.The combined effects of hemodynamic changes and the destructive effectsof macrophages through their release of proteolytic enzymes may lead todevelopment of the defect.

Scanning electron microscopy studies of vascular corrosion casts of theACA-OA bifurcation and double immunostaining of the vascular wallillustrates that normal endothelial cells are seen at the apical intimalpad, and the endothelial cell markings are elongated in the direction ofthe blood flow. The endothelial and the smooth-muscle layer form twocontinuous layers. In stage I, there are roughened apical intimal padwith irregularly shaped imprints, and loss of eNOS expression at theapical intimal pad is observed. In stage IIA, there is shallow elevationsurrounded by an area of depression of the apical intimal pad. Swellingof the vessel wall at the apical intimal pad is shown, and part of thisswollen area lacks eNOS expression and shows subendothelial expressionof α-SMA-positive cells. In stage IIB, there is pyramid-shaped elevationof the apical intimal pad, and the surface of this elevation is coveredby abnormal imprints. Thinning and degradation of the smooth-musclelayer creates a defect in the inflammatory zone (arrow) and producesvessel wall protrusion. In stage III, there is saccular aneurysm coveredwith abnormal imprints, expansion of the inflammatory zone defect, anddestruction and protrusion of the vessel wall representing the nidus ofthe cerebral aneurysm.

Stage IIA has early inflammatory changes characterized by SMC migrationand macrophage infiltration. A sagittal cut of the left ACA-OAbifurcation viewed at low and high magnification shows swelling of theapical intimal pad. Double immunostaining of an ACA-OA section with eNOSantibodies and α-SMA shows swelling of the vessel wall at the apicalintimal pad; part of this swollen area lacks eNOS expression and showsmigration of α-SMA-positive cells into the neointima. In tripleimmunostaining of an ACA-OA section with antibodies against eNOS, α-SMA,and macrophages, macrophage expression confirms the presence of aninflammatory zone.

BRIEF SUMMARY OF THE INVENTION

The present invention generally concerns a method for detection ofaneurysms, including cerebral aneurysm, using non-invasive molecularimaging techniques, and in particular cases further concerns treatmentof the aneurysm.

The present invention can detect any aneurysm, regardless of the stageor type of the aneurysm. In early stage aneurysms, the initial changesare characterized by the alteration of the endothelium andsubendothelium and migration of medial smooth cells in the intima. Atthe late stage of degeneration, there is a disappearance of the media,and the aneurysmal wall just consists in an endothelium and a few fibersof collagen (adventicia). However, because these different stages ofdegeneration coexist in the same aneurysm, the invention can detect anyaneurysm.

In particular aspects of the invention, the methods and compositionsconcern targeting a specific component of the subendothelium at theaneurysm. In certain cases, the specific component in the subendotheliumthat is targeted by compositions of the invention for the detection ofaneurysmal lesions are migrating SMCs that are in a contractilephenotype, which is in contrast to SMCs in neointima formation that arein a synthetic phenotype. That is, specific markers of SMC phenotypewill allow one to differentiate SMCs in aneurysmal wall from SMCs inneointima. In specific embodiments, particular proteins on the surfaceof SMCs unique to contractile SMCs are the targets fordetection/treatment of the aneurysm. In specific embodiments, integrinreceptors can serve as markers of SMC phenotype. In further specificembodiments, the differentiated contractile SMCs comprise α1β1 integrin(which is a receptor for laminin, collagen I, and collagen IV) and α7β1integrin, which is a receptor for laminin-1. However, in the arterialwall, α1β1 integrin is expressed on SMCs but also on macrophages (whichinvade the arterial wall in pathological conditions such asatherosclerosis and aneurysm). Therefore, in some embodiments α7β1 istargeted, because in the arterial wall it is expressed exclusively bySMCs. α7 integrin expression confers a gain of function-motile phenotypeto immobile cells and is responsible for transduction of thelaminin-induced cell motility, in certain aspects of the invention.Laminin-1 is also useful in the invention as a marker of aneurysmallesion, because it is the specific ligand of α7 integrin, and α7integrin mediates adhesion and migration of SMCs on laminin-1.

Thus, methods of the present invention are based at least in part on twocharacteristic features of early aneurysmal lesions: 1) the degenerationof endothelium and subendothelium that exposes underlying components;and 2) the migration in the subendothelium of SMCs of contractile,migrating phenotype (α7 integrin positive cells that bind specificallylaminin-1). In specific cases, the method employs a labeled antibodydirected against α7 integrin or laminin-1, for example, and the labeledantibody will have such characteristics that it binds exclusively toSMCs exposed at the luminal surface of the vessel, as opposed to beingwithin the media. That is, such labeled antibody will not be able tobind contractile SMCs in the medial layer of normal arterial wall. Inparticular cases, the labeled antibody is coupled to a macromolecularcompound (such as Dextran, for example) that keeps the compound in theintravascular compartment but yet allows it to be cleared from theintravascular compartment. Such a compound will bind exclusively SMCsexposed at the luminal surface of the vessel due to endothelial andsubendothelial degeneration but will not be able to bind contractileSMCs in the medial layer of normal arterial wall. In vivo detection ofthe aneurysm is achieved, and the method may use different molecularimaging techniques such as immunoscintigraphy using antibodyradiolabeled with 99mTc-dextran or MRI using antibody conjugated toGadolinium-DTPA-dextran, for example.

In one embodiment of the invention, there is an isolated composition,comprising a cell targeting molecule, an intravascular targetingmolecule, and a label. In a specific embodiment, the composition furthercomprises a therapeutic agent. In a further specific embodiment, thecell targeting molecule is an antibody or a peptide. In specificembodiments, the antibody immunologically reacts with an integrinreceptor on the cell surface of smooth muscle cells or a laminin (suchas laminin 1). In certain aspects, the integrin receptor is α1β1, α7β1,or α8β1. In particular embodiments, the intravascular targeting moleculeis a polymer, such as one selected from the group consisting of dextran,albumin, transferrin, globulins, pectin, gelatin, and cellulosederivatives. In some cases, the label is a radionuclide, a fluorophore,a lucigen, or a paramagnetic chelator or microbubble contrast agent. Infurther embodiments, the therapeutic agent is selected from the groupconsisting of a thrombogenic molecule, a polymerisable molecule,fibrinogen, a cell matrix protein (such as elastin, fibronectin, orlaminin), a synthetic peptide, a cell growth factor, an elastaseinhibitor, and a MMP inhibitor. In particular cases, the composition iscomprised in a pharmaceutically acceptable excipient.

In another embodiment of the invention, there is a method of detectingand/or treating a cerebral aneurysm in an individual, comprising thestep of delivering an effective amount of a compound of the invention tothe individual. The individual may be at risk for developing ananeurysm, has a history of cerebral aneurysm, or is asymptomatic with nohistory or known risk of cerebral aneurysm (method may be used as a massdetection or routine screen for individuals). In specific cases, thecomposition is delivered to the individual once or more than once. Incertain aspects, the individual is provided an additional therapy foraneurysm, such as one that comprises medication, surgery, endovascularcoiling, or a combination thereof.

In another embodiment of the invention, there is a kit for detectionand/or treatment of aneurysm, comprising the compound of the invention,housed in a suitable container. In specific embodiments, the kit furthercomprises a therapeutic agent.

In general embodiments of the invention, one may be able todifferentiate individuals in an “at-risk” aneurysm sub-group within acerebral aneurysm population.

In particular aspects of the invention, α1β1, α7β1, α3β1, α8β1integrins, and/or laminin 1 are indicators that there is a risk for ananeurysm to rupture; in specific embodiments,one can utilize one or moreof these targets to detect the cerebral aneurysms, that are prone torupture.

In other specific embodiments, the α7 expression, or the ratio ofexpression of α7/α5 is a marker of aneurysm that is “at risk” ofrupture.

The foregoing has outlined rather broadly the features and technicaladvantages of the present invention in order that the detaileddescription of the invention that follows may be better understood.Additional features and advantages of the invention will be describedhereinafter which form the subject of the claims of the invention. Itshould be appreciated by those skilled in the art that the conceptionand specific embodiment disclosed may be readily utilized as a basis formodifying or designing other structures for carrying out the samepurposes of the present invention. It should also be realized by thoseskilled in the art that such equivalent constructions do not depart fromthe spirit and scope of the invention as set forth in the appendedclaims. The novel features which are believed to be characteristic ofthe invention, both as to its organization and method of operation,together with further objects and advantages will be better understoodfrom the following description when considered in connection with theaccompanying figures. It is to be expressly understood, however, thateach of the figures is provided for the purpose of illustration anddescription only and is not intended as a definition of the limits ofthe present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an exemplary schema outlining embodiments of the presentinvention for cerebral aneurysm formation versus neointima formation.

FIG. 2 is an exemplary schema outlining embodiments of the presentinvention regarding treatment of neo-intima formation vs. cerebralaneurysm.

DETAILED DESCRIPTION OF THE INVENTION

This application incorporates WO 2007/092419 by reference herein in itsentirety.

Unless defined otherwise, technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. For purposes of the presentinvention, the following terms are defined below.

As used herein, the use of the word “a” or “an” when used in conjunctionwith the term “comprising” in the claims and/or the specification maymean “one,” but it is also consistent with the meaning of “one or more,”“at least one,” and “one or more than one.” Some embodiments of theinvention may consist of or consist essentially of one or more elements,method steps, and/or methods of the invention. It is contemplated thatany method or composition described herein can be implemented withrespect to any other method or composition described herein.

I. Definitions

The term “aneurysm” as used herein refers to an abnormal bulge or“ballooning” in the wall of an artery.

The term “cerebral aneurysm” as used herein refers to a cerebrovasculardisorder wherein a localized weakness in the wall of a cerebral bloodvessel (artery) results in a localized dilation or ballooning of theblood vessel. The majority of aneurysms are saccular in shape. It mayalso be referred to as a Saccular Cerebral Arterial Aneurysm (SCAA) orintracranial aneurysm.

The term “effective amount” or “therapeutically effective amount” asused herein is defined as an amount of the agent that will decrease,reduce, inhibit or otherwise abrogate an aneurysm. Thus, an effectiveamount is an amount sufficient to detectably and repeatedly ameliorate,reduce, minimize or limit the extent of the disease or at least one ofits symptoms.

As used herein, the term “tunica intima” (or just intima) comprises alayer of endothelial cells supported by a basement membrane and aninternal elastic lamina. The connective tissue between the endotheliumand the internal elastic lamina is called “subendothelium.” The internalelastic lamina (I.E.L) is part of the intima.

As used herein, the “subendothelium” is a definite zone within theintima and corresponds to the connective tissue between endothelial celllayer and inner elastic lamina.

As used herein, the “tunica media” refers to the main layer of theartery wall and comprises SMCs.

The “tunica adventitia” as used herein refers to the outermost layer andcomprises mostly collagen. Cerebral arteries do not have the externalelastic lamina unlike systemic arteries.

II. General Embodiments of the Invention

Intracranial aneurysms are a major public health problem; it isestimated that approximately 5% of the population harbors an unrupturedintracranial aneurysm (Iwamoto et al., 1999). The consequences ofrupture are catastrophic: approximately 50% of patients die during thefirst post-rupture month, and 60% of deaths occur within 2 days of theonset of aneurismal sub-arachnoid hemorrhage (SAH) (Broderick et al.,1994). Half of the survivors manifest physical or psychosocial deficits1 year after SAH (Hackett and Anderson, 2000). Clip placement and coilocclusion to treat ruptured intracranial aneurysms, aimed at avoidingrecurrent bleeding, have no direct effect on the recovery from theinitial hemorrhage.

For surgical clipping, after a craniotomy, the surgeon then spreads thebrain tissue apart, opens the subarachnoid cisterns under operativemicroscope and then places a tiny metal clip across the neck to stopblood flow into the aneurysm and exclude it from the blood stream.Endovascular treatment of brain aneurysms is a minimally invasiveprocedure which requires insertion of a catheter into the femoral arteryand navigating it through the vascular system into the aneurysm(referred to as Endovascular Coiling (Guglielmi detachable coil)). Tinyplatinum coils are threaded through the catheter and deployed into theaneurysm, blocking blood flow into the aneurysm and preventing rupture.However, these treatments are responsible for 15% of the morbidity andmortality. Recurrence is not insignificant: 2.2% at 10 years and 9.0% at20 years (Molyneux et al., 2002). Given the poor prognosis of rupturedintracranial aneurysms, cerebral aneurysms should be detected beforerupture, however current diagnosis means are invasive and expensive(e.g., 4-axle digital subtraction angiography), and no mass detectioncan be currently considered. Therefore, there is a need for developingnon-invasive diagnosis and safer treatment for intracranial aneurysms.

Preliminary data from the literature have suggested that degeneration ofthe endothelium and subendothelium at specific site on the bifurcationof cerebral arteries is a characteristic feature of cerebral aneurysmformation. In the present invention, these degenerative changes renderunderlying components of the arterial wall abnormally expressed at theluminal surface of the artery, and these components are a useful atarget for in vivo intravascular immuno-detection of cerebral aneurysms.The diagnosis of aneurismal lesions is made by coupling a specificantibody directed against a subendothelial component of the arterialwall to a label moiety, in certain cases. In specific embodiments, thelabeled antibody is coupled to a compound (such as dextran) thatconfines it into the intravascular compartment, yet allows it to berapidly cleared from the intravascular space. This method allows thediagnosis of cerebral aneurysm at an early stage, rendering possible abiological treatment for repairing the wall before irreversible damages.The treatment uses the same specific antibody coupled to a therapeuticmoiety. Several therapeutic agents have already been considered such asthe use of a thrombogenic or polymerisable molecule (intended to clogthe aneurysms) or proteins (elastin, fibronectin, or fibrinogen, forexample), or cell growth factors (intended to reinforce or make thefundus thicker and stronger). Thus, an antigenic component of thesubendothelium of the arterial wall is useful for in vivoimmuno-detection of early-stage aneurysm, although any stage of aneurysmmay be detected and treated with the present invention.

III. Cerebral Aneurysm

The present invention is useful for detection and treatment of anycerebral aneurysm. Cerebral aneurysms are classified by size and shape,with small aneurysms having a diameter of less than 15 mm and largeraneurysms are those classified as large (15 to 25 mm), giant (25 to 50mm), and super giant (over 50 mm). Saccular aneurysms, the most commonform, have a saccular outpouching, whereas berry aneurysms areparticular saccular aneurysms having necks or stems resembling a berry.Fusiform aneurysms lack stems.

Cerebral aneurysms commonly occur on the arteries at the base of thebrain, known as the Circle of Willis. About 85% of cerebral aneurysmsdevelop in the anterior part of the Circle of Willis, thereby involvingthe internal carotid arteries and their major branches that supply theanterior and middle sections of the brain. Common locations are asfollows: the anterior communicating artery (30-35%), the bifurcation ofthe internal carotid and posterior communicating artery (30-35%), thebifurcation of the middle cerebral artery (20%), the bifurcation of thebasilar artery, and the remaining posterior circulation arteries (5%).

Cerebral aneurysms may occur at any age, although they are more commonin adults than children, and more common in women than men.

Most of intracranial aneurysms are clinically quiescent until theyrupture. Onset of the aneurysm usually is sudden, with no warning.Rupture of a cerebral aneurysm often results in bleeding into thesubarachnoid space or the brain itself, resulting in a subarachnoidhemorrhage (SAH) or intracranial hematoma (ICH) (Ruptured aneurysm).Although unruptured aneurysms are usually asymptomatic, some may beknown either because they are multiple (The 4-axle digital subtractionangiography may reveal multiple unruptured aneurysms), or symptomatic(third cranial nerve palsy, headache, orbital pain) or incidental (brainimaging performed for a neurological non-aneurysmal disease)

The histology of normal cerebral artery junction is described asfollows: the intimal pad is just distal to apex on distal side of ACA,and the pad is composed of spindle-shaped cells similar to the medialsmooth muscle cells; the internal elastic lamina is continuous along thecurvature of the apex, but at the proximal margin of the intimal pad, itis split into several layers and considerably fragmented under theintimal pad, and just distal to the intimal pad, it is thinned andfragmented for a short distance; no medial defect is found. In earlystage aneurysms, initial changes are localized almost exclusively at theintimal pad and its neighboring distal portion; the wall does notsignificantly protrude; and there is fragmentation of internal elasticlamina and slight thinning of the smooth muscles cells layer. Inadvanced stage aneurysms, there is complete disappearance of theinternal elastic lamina at the level of the aneurysmal neck, the medialayer (SMC) ceases abruptly proximal to the neck, and the aneurysmalwall consists only of a fibrous adventitia and a layer of endothelialcells. Aneurysms form when hemodynamic stress plus pulsatile flowpatterns initiate degenerative changes in the endothelial layer adjacentto the apex (distal side) of the arterial bifurcation, and theseendothelial injuries are followed by degenerative changes in theinternal elastic lamina, then in the medial layer (affecting the SMCs).

IV. Risk Factors for Rupture of Aneurysm

After an aneurysmal subarachnoid hemorrhage, nearly half of the patientsdie, with the remaining half who survive suffering from irreversiblecerebral damage. More unruptured cerebral aneurysms are identified withincreasing use of noninvasive neuro-imaging techniques (for example,magnetic resonance and computerized tomography angiography). The risk ofrupture in aneurysms smaller than 10 mm is a 0.5 to 2% annual risk, inspecific embodiments. Growing aneurysms and those larger than 10 mm runa higher risk for rate of rupture.

Risk factors for aneurysm rupture include the size of aneurysms instable compared With Growing Lesions. In the ISUIA (ISUIA=InternationalStudy of Unruptured Intracranial Aneurysms) (1998) it has been pointedout that the size and location of aneurysms were independent predictorsof rupture. In Group 1, aneurysms that were 25 mm or more in diameterhad a rupture rate of 6% in the 1st year. It was observed that aneurysmsthat ruptured at a later time had more often increased significantly insize (> or =1 mm) than the largest aneurysms in patients withoutbleeding (Juvela, 1993, 2002, and 2000).

Locations of aneurysms may also be a factor. For example, aneurysms ofthe vertebrobasilar and middle cerebral arteries have a statisticallyhigher probability of subsequent bleeding. In ISUIA Group 1, forexample, the relative risk of rupture was 13.8 for aneurysms that werelocated at the basilar tip, whereas the relative risk was 13.6 for thosein the vertebrobasilar or posterior cerebral artery distribution,compared to other locations. For posterior communicating arteryaneurysms, the relative risk of rupture was 8.0. The relative risk ofrupture was 5.1 for aneurysms at the basilar tip in Group 2.

The shape of the aneurysms has an impact on rupture, in specificexamples. For example, multilobed lesions have a significantly higherrisk of hemorrhage than do single-lobed unruptured aneurysms. In someembodiments, the age and sex of the individual is a factor; females havea risk factor affecting both aneurysm formation and growth, for example.In other cases, cigarette smoking hastens the growth of preexistinganeurysms (Juvela, 2002).

Families having intracranial aneurysms and rupture history have agreater risk for rupture. In families with two or more first-degreemembers, especially siblings and mother-daughter pairs, or two first-and second-degree members with SAH, the risk that other relatives willharbor unruptured intracranial aneurysms is approximately 9 to 11%,which is higher than in the general population (Raaymakers et al., 1998;Ronkainen et al., 1997)

Finally, genetic conditions in some cases have an impact on risk factorsfor aneurysm rupture. For example, the presence of ADPKD(ADPKD=autosomal-dominant polycystic kidney disease) is associated witha 15% prevalence (Rinkel et al., 1998) of cerebral aneurysms.Individuals having Type IV Ehlers-Danlos syndrome, hereditaryhemorrhagic telangiectasia, neurofibromatosis Type 1,alpha-1-antitrypsin deficiency, Klinefelter syndrome, tuberoussclerosis, Noonan syndrome, or alpha-glucosidase deficiency have apropensity for intracranial aneurysms compared with the generalpopulation, in certain embodiments of the invention.

V. Exemplary Molecular Basis of the Invention

Due to the degeneration of the endothelium and subendothelium of theaneurysmal wall, SMCs that migrate into the intima are abnormallyexposed at the luminal surface of the arterial wall and become availableto react with labeled antibodies. These cells can be labeled in vivousing a cell surface marker (cytoskeletal antigen (such as α-SMA) areprotected from extracellular fluid by the cellular membrane and can notbe labeled in vivo).

The present invention exploits the pathogenesis of cerebral aneurysm inwhich smooth muscle cells (SMCs) have a critical role. Therein, one ormore cell surface markers of SMCs, for example specific integrinreceptors, are antigenic components in the arterial wall targeted for invivo immuno-detection of aneurysm.

A. Background on Smooth Muscle Cells (SMCs) and Their Role in ArterialWall Repair After Injury

1. Smooth Muscle Cell (SMC)

SMC is the sole cell type normally found in the media of mammalianarteries. In the adult, it is a terminally differentiated cell thatexpresses cytoskeletal marker proteins like smooth muscle alpha-actin(α-SM actin) and smooth muscle myosin heavy chain (SMMHC), and contractsin response to chemical and mechanical stimuli. They take part in thecontrol of blood pressure and flow; at this stage they are referred toas being in a contractile phenotype. However, the smooth muscle cell isable to revert to a proliferative and secretory active state equivalentto that seen during vasculogenesis in the fetus; at this stage they arereferred to as being in a synthetic phenotype. SMCs in their syntheticphenotype have a fibroblast-like appearance, a prominent endoplasmicreticulum and Golgi complex, few filaments and only a weak reactivityfor α-SM actin. They secrete extracellular matrix components: laminin,fibronectin, collagen and elastin (Thyberg et al., 1997;Hultgardh-Nilsson et al., 1997). The transition from a contractile to asynthetic phenotype occurs in vascular diseases such as atherosclerosisand restenosis after angioplasty. In these diseases, in response toendothelial injury, smooth muscle cells migrate from the media to theintima, they dedifferentiate into a synthetic phenotype, proliferate andsecrete components of the extracellular matrix and form what is called aneointima or myo-intimal hyperplasia or intimal thickening (Campbell andCampbell, 1985; Schwartz and Reidy, 1987). Neointima formation is acommon mechanism of arterial wall repair after endothelial injury

2. Factors that Control SMC Phenotype

Thyberg et al. (1997) have made extensive research on the control of SMCphenotype. They demonstrated first that laminin promotes the expressionof a differentiated smooth muscle phenotype in vitro and in vivo,whereas fibronectin stimulates the cells to adopt a synthetic phenotype.Then, they demonstrated that after being converted into a syntheticphenotype, the cells do not start to proliferate without exogenousmitogen stimulation. They showed that some growth factors and especiallyplatelet-derived growth factor (PDGF) stimulates SMC proliferation.After stimulation with PDGF, converted SMCs divide and produce their ownPDGF which stimulates their growth in an autocrine and paracrine manner(Sjolund et al., 1988). Therefore, it is concluded that at least tworequirements need to be fulfilled for inducing the synthetic,proliferating phenotype of SMCs:

First, the cells must adhere to a substrate of fibronectin and second,they must be stimulated with growth factors and especially PDGF. Thesestudies are in agreement with others demonstrating that there is anaccumulation of fibronectin at the site of arterial injury, inassociation with neointima formation (Bauters et al., 1995; Chemnitz andCollatz Christensen, 1983) suggesting an important pathophysiologicalrole of fibronectin in neointima formation and vascular wall repair(Hedin and Johan, 1987; Boudreau et al., 1991; Molossi et al., 1995). Tomodulate the phenotype of SMCs, laminin and fibronectin bind to integrinreceptors on the surface of SMCs.

3. Integrin Receptors on SMCs

Integrins are a family of receptors involved in cell interactions withextracellular matrix (ECM) components and with other cells. Eachintegrin receptor is a heterodimer in which one of several homologous αsubunits associates noncovalently with a β subunit. Some integrinsubunit combinations recognize multiple ligands, while others arerelatively specific. Although some integrins are widely expressed by avariety of cell types, others have a restricted distribution.

a. Integrin Receptors for Laminin

Several integrins (α1β1, α2β1, α3β1, α6β1, α7β1 and αvβ3) bind laminin(Clyman et al., 1994). It has been demonstrated that only α1β1, α3β1,α7β1 and αvβ3 are expressed on human SMCs in vivo. α2β1 is a receptorfor collagen I to VI and laminin. Despite the potentially significant invitro functions of α2β1 in modulating SMC behavior, studies were unableto detect this integrin complex in normal or atherosclerotic humanarteries (Glukhova et al., 1993). α2β1 has not been detected in SMCs invivo (Thorsteinsdottir et al., 1995).

α1β1: is a receptor for laminin, collagen-I, collagen IV. Human medialSMCs (which in vivo are surrounded by a basement membrane that containslaminin-1 and/or laminin 3 and collagen IV) express high level of α1β1.α1 subunit expression is an exceptional feature of SMCs. Other celltypes (fibroblasts, endothelial cells, keratinocytes, striated muscles,and platelets) contained trace amounts of α1β1 integrin (Belkin et al.,1990). Only activated T cells, monocytes also express α1β1 integrin.α1β1 integrin expression is characteristic of differentiated SMCs. Ithas been demonstrated that SMCs from intimal thickening of human adultaorta express less α1 subunit of α1β1 integrin than SMCs from adultaortic media (Belkin et al., 1990). In contrast, it has been reported ina rat vascular injury model that α1β1 is expressed by intimal SMC inresponse to vascular injury (46). This discrepancy may be explained bythe species-specificity of integrins.

α3β1: is able to bind a variety of ECM components including laminin,nidogen/entactin, fibronectin, and collagen I. It is expressed in vivoin medial SMC of human artery (Hillis et al., 1998). It is alsoexpressed on B-lymphocytes and cells of kidney glomerulus.

α7β1: is a specific receptor for laminin-1. This integrin has a highlytissue-specific and limited expression pattern. It is a muscle specificintegrin being expressed by all major types of muscle tissue, includingskeletal, cardiac and smooth muscle. Its presence in all muscle typessuggests a role for this integrin in transducing myofilament-generatedforces to anchoring sites in the surrounding laminin-rich basementmembrane during cellular contractile activity. No studies havedocumented the expression of α7β1 in human vascular SMCs. But, it hasbeen demonstrated that murine vascular SMCs express the α7 integrinreceptor. The expression of α7 integrin in SMCs is associated with theirdifferentiated phenotype and mediates their interaction with laminin(Yao et al., 1997). Studies have demonstrated that α7 integrinexpression confers a gain of function-motile phenotype to immobile cellsand may be responsible for transduction of the laminin-induced cellmotility (Echtermeyer et al., 1996). Therefore, in embodiments of theinvention, there is a role of α7 integrin in migration of SMCs to theintima after vascular injury. It is likely that during neointimaformation, highly differentiated SMCs, which were originally arranged inconcentric layers and encircled by basement membranes, become motile andmigrate into the intima toward growth factor signals usinglaminin-binding α7 integrin. In the intima, growth factors (especiallyPDGF) and extracellular matrix (fibronectin) modulate SMC phenotype andintegrin expression (switching from α7 to α5β1 integrin expression)leading to the formation of a neointima. Furthermore, a recent study hasdemonstrated the importance of α7 integrin in vascular remodeling(Welser et al., 2007). Using a carotid ligation animal model, theauthors have found a profound increase in vascular remodeling andneointima formation in the carotid arteries of α7 integrin-null micesubjected to ligation.

αvβ3: is able to bind a variety of ECM components including laminin,vitronectin, von Willebrand factor, thrombospondin, osteopontin,fibrinogen and fibronectin. It is expressed on different cell typesincluding platelets, endothelial cells and SMCs. Studies havedemonstrated that αvβ3 is expressed on SMCs in the media of normal aswell as atherosclerotic coronary artery and on SMCs in the neointima.αaβ3 was also strongly expressed by luminal endothelium (Hoshiga et al.,1995). Clinical studies have shown that c7E3, an antibody directedagainst β3 integrin reduces SMC migration and neointima formation and isuseful in the prevention of the SMC response in restenosis afterangioplasty (Topol et al., 1997).

b. Integrin Receptors for Fibronectin

The integrin receptors for fibronectin are α3β1, α4β1, α5β1, α8β1, αvβ1,αvβ3, α-IIb/β. α4β1, α5β1, α8β1, αvβ1 are specific receptor forfibronectin (Topol et al., 1997).

α5β1: is a specific receptor for fibronectin. This integrin has beenfound to be expressed by SMCs in the media of human aorta whereas thisprotein was absent in the destructive media of aneurysmal aorta. Themarked decrease in integrin α5β1 correlated to a decrease in density ofSMCs (Cheuk and Cheng, 2004). Studies in a rat model of vascular injury,have shown that α5β1 integrin was not expressed in medial SMCs buthighly expressed after vascular injury by the less differentiated SMCsat the luminal surface of the neointima. This subpopulation of α5β1positive SMCs repairs the arterial wall by assembling the fibronectinmatrix. Soluble fibronectin protomers polymerize on the surface of theseα5β1 positive cells. This assembly process is of paramount importancefor wall repair because only insoluble fibrillar fibronectin can act asan adhesive ligand and regulate cell function (Pickering et al., 2000).In conclusion, these data indicate that α5β1 integrin, a specificreceptor for fibronectin is critical for maintaining the integrity ofthe medial layer of normal artery and for the processus of wall repairand neointima formation after vascular injury. It is also expressed by Tcells, monocytes, platelets.

α8β1: is a specific receptor for fibronectin. α8 subunit has arestricted cellular distribution. It is expressed in vascular andvisceral smooth muscle cells. SMCs of arteries show an intense staining.The endothelial cells of vessels do not stain. The cells that expressedα8 subunit function as contractile cells (Schnapp et al., 1995).

c. Integrin Receptors as Markers of Smooth Muscle Cell Phenotype

According to the above data, α1β1, α7β1, α3β1, α8β1 are useful specificmarkers of differentiated contractile SMCs (these integrins are highlyspecific of SMCs and are not expressed by other cell types such asendothelial cells or platelets). Moreover, α7 integrin-expressionconfers a gain of function-motile phenotype to immobile cells and may beresponsible for transduction of the laminin-induced cell motility.

In contrast, α5β1 a specific receptor or fibronectin is expressed byless differentiated SMCs of the neointima formation and is critical forfibronectin assembly and wall repair after injury. αvβ3 is an integrinthat is widely expressed in the vessel wall (endothelial and SMCs cells)and has multiple ECM ligands is overexpressed in neointima formation.

d. Neointima Formation in Occlusive Arterial Disease.

Neointima formation is a common mechanism of arterial wall repair afterendothelial injury and occurs in vascular diseases such asatherosclerosis and restenosis after angioplasty. Endothelium injuryinduces platelet adhesion which induces the release of growth factorsand especially PDGF. PDGF stimulates migration of SMCs for the media tothe intima. Within the intima, SMCs bind fibronectin (FN) of thebasement membrane. Fibronectin stimulates the cells to adopt a syntheticphenotype and then PDGF stimulate the proliferation of thededifferentiate SMCs. SMCs in the neointima highly expressed α5β1 andαvβ3 integrins.

B. Exemplary Model for Pathogenesis of Cerebral Aneurysm

It has been demonstrated that hemodynamic stress is responsible forendothelium injury. But, in contrast to vascular diseases such asatherosclerosis and restenosis after angioplasty, neointima formationdoes not occur and there is no repair of the arterial wall. Duringaneurysm formation, there is disappearance of the medial layer. At anadvanced stage, the aneurysm wall consists only of a fibrous adventitiaand a layer of endothelial cells. Therefore, some factors thatcontribute to wound healing may be missing in the course of cerebralaneurysm formation. As described above, studies have demonstrated thatfibronectin (as well as collagen IV and I) normally expressed in thesubendothelium of artery, disappears in early aneurysmal lesions. Thisdegeneration of the endothelial basement membrane and the subendothelialconnective tissue may be due to endothelial dysfunction. Indeed,degeneration of endothelial cells in aneurismal walls has beendemonstrated by scanning electron microscopy (described above) and thesedegenerated cells may decrease the production of ECM. Also, hemodynamicstress by itself may alter the subendothelium matrix. The absence offibronectin in aneurysm wall is a critical feature in aneurysm formationconsidering the role of this ECM protein in wound repair and its role inmodulation of SMC phenotype (as described above). In the invention, SMCsmigrate from the media to the intima toward growth factor (especiallyPDGF) secreted after endothelial injury using laminin-binding α7integrin. Because of the absence of fibronectin, SMCs can not switchtheir phenotype from a contractile to a synthetic phenotype. These cellscan not proliferate to form a neointima. Furthermore, since fibronectinis known to facilitate SMC survival by providing integrin-mediatedinhibition of apoptose, in absence of fibronectin, SMCs may enterapoptosis and disappear. The SMCs that migrate to the intima in theearly stage of aneurysm formation are SMCs of contractile phenotype andexpressed α1β1, α3β1, α7β1 and α8β1 integrins in contrast to SMCs in theneointima formation which are of synthetic phenotype and express α5β1and αvβ3 integrins.

FIG. 1 illustrates an exemplary schema outlining embodiments of thepresent invention for cerebral aneurysm formation versus neointimaformation. In certain embodiments of the invention, integrin α7β1 whichis specific cell surface marker of SMCs (they are not expressed byendothelial cells nor platelets) and is characteristic of theircontractile phenotype is the best antigenic subendothelial components inthe arterial wall useful for in vivo immuno-detection of early-stageaneurysm. Data from the literature suggest a prominent role for β7integrin in vascular remodeling as described above, and in specificembodiments β7 integrin is a useful target antigen.

In vivo, human medial contractile SMCs are surrounded by a basementmembrane that contains laminin 1 and/or laminin 3 (Yao et al., 1997)Laminin 1 is not expressed by the basement membrane of endothelium(which is composed of laminin 8 and 10) (Halmann et al., 2005; Falk etal., 1999; Viranen et al., 2000). It has been demonstrated that α7integrin binds to specific laminin isoforms mediating adhesion andmigration of SMCs (Yao et al., 1996): α7 integrin binds to laminin 1 anda mixture of laminin 2 and 4 but not to laminin 5. In contrast, it hasbeen demonstrated that Laminin 5, an ECM protein found predominantly inepithelial tissues is expressed at low-level in the intima of normalvessels but is overexpressed in the neointima of injured vessels(Kingsley et al., 2002). In specific aspects, laminin 1 is an antigenicsubendothelial component of aneurismal lesions while laminin 5 is amarker of neointima formation.

In certain aspects of the invention, immunohistochemistry is utilized tocharacterize the in situ expression of α7 integrin and laminin-1 in thearterial wall of early aneurysmal lesions in an established animal modelof cerebral aneurysm. In other aspects of the invention, the in vivodetection of cerebral aneurysms is characterized by nuclear imaging ormolecular MRI using labeled antibodies directed against α7 integrin orlaminin-1 injected intravascularly.

VI. Compounds of the Invention

The compounds of the invention (which may also be referred to ascompositions of the invention) are prepared such that they are suitablefor detection and/or therapy of an aneurysm in an individual. In generalembodiments, the compounds comprise different combinations of atargeting molecule, a label, an intravascular targeting molecule, and atherapeutic agent.

A. Cell Targeting Molecule

The cell targeting molecule of the invention is any moiety that issuitable to target a composition to a smooth muscle cell at a cerebralaneurysm. In specific embodiments, the targeting molecule is an antibodyor a peptide, and in particular cases the antibody or peptide targetsproteins on the surface of the smooth muscle cell, such as proteins thatare receptors, for example.

1. Antibodies

In specific embodiments of the invention, the cell targeting molecule isan antibody. Although the antibody may immunologically react with anytarget that identifies a smooth muscle cell at the site of an aneurysm,in specific embodiments the antibody targets a protein, such as areceptor, on the cell. In certain cases, the antibody will bindexclusively smooth muscle cells exposed at the luminal surface of thevessel, and in particular cases the antibody does not bind contractilesmooth muscle cells in the medial layer of the normal arterial wall. Inspecific embodiment, the receptor is an integrin or is a laminin. Infurther specific embodiments, the antibody recognizes α7 integrin orlaminin-1

2. Peptides

In other embodiments of the invention, the cell targeting molecule is apeptide. Although the peptide may bind with any target on a smoothmuscle cell at the site of an aneurysm, in specific embodiments thepeptide targets a protein, such as a receptor, on the cell. Inparticular cases, the peptide will bind exclusively smooth muscle cellsexposed at the luminal surface of the vessel, and in specific cases thepeptide does not bind contractile smooth muscle cells in the mediallayer of the normal arterial wall. In specific embodiment, the receptoris an integrin or is a laminin. In further specific embodiments, thepeptide recognizes α7 integrin or laminin-1. In further specificembodiments, the peptide also known as peptidomimetic (smallprotein-like chain design to mimic a peptide) competes with laminin-1 tobind α7 integrin. This peptidomimetic of laminin-1 may be derived fromthe G domain of the α1 chain of laminin-1 such as the α1 chainaminoacids 2179-2198 “SN peptide” (Khan et al., 2002)

B. Labels

In particular aspects of the invention, one or more labels are comprisedon a composition of the invention. The label may be attached to the celltargeting molecule, the intravascular targeting molecule, and/or thetherapeutic agent. Although any labels suitable in the art may beemployed, in specific embodiments the label is a radionuclide, afluorophore, a lucigen, or a paramagnetic chelator or microbubblecontrast agent.

A label moiety may be attached to the antibody moiety using techniquesreadily available to the public. The label moiety may be a radioactivelabel such as a gamma ray emitting radionuclide: 111 Indium,Technetium-99m, iodine 123 (123I), iodine 125 (125I). A chelating agentsuch as dipropylaminetetraacetic acid (DPTA) may be used to associatethe radioactive label to the antibody. The label moiety may be apositron emitting radionuclide such as Fluorin-18, carbon-11, Gallium68. The label moiety may be a near infrared fluorophore (near infraredfluorescent dye) such as Cy7-NHS, Cy5.5 (Amersham Pharmacia). The labelmoiety may be a MRI contrast agent, such as paramagnetic lanthanideGadolinium or superparamagnetic particles of iron oxide (SPIOs), forexample. The label moiety may be also a microbubble contrast agent.

C. Intravascular Targeting Molecule

In general embodiments of the invention, the composition comprises amoiety that prevents the cell targeting molecule from moving beyond theluminal surface of the aneurysmal wall to bind smooth muscle cells inthe medial layer of normal vascular wall that harbors an intactendothelium. Any macromolecular agent with intravascular retention maybe coupled to the cell targeting molecule is useful. The molecule mayhave a molecular weight that confines it within the vessels, yet allowsit to be cleared from the intravascular compartment. In a specificembodiment, the molecule is a macromolecule, and macromolecular speciesmay include any molecule, natural or synthetic, which has a molecularweight in excess of 1 kilodalton such as but not limited to albumin,transferrin, globulins, pectin, gelatin, dextran, and cellulosederivatives, for example.

In specific embodiments, the molecule is a polymer. In particularembodiments, the molecule is dextran. Dextran is a hydrophilic moleculethat does not pass the phospholipid bilayer of endothelial membrane.Antibody (or any cell targeting molecule) conjugated to dextran will notpass the endothelium. The conjugate will bind specifically smooth musclecells at the luminal surface of the aneurysmal wall, such as α7 integrinor laminin-1 exposed at the luminal surface of the aneurysmal wall, andcannot bind α7 integrin or laminin-1 on SMCs in the medial layer ofnormal vascular wall that harbors an intact endothelium.

D. Therapeutic Agent

In particular aspects of the invention, the compounds for delivery tothe individual with the aneurysm or suspected of having an aneurysmcomprise one or more therapeutic agents. In specific cases, thetherapeutic agent is bound directly to the label, the intravasculartargeting molecule, and/or the cell targeting molecule. Although inspecific cases the therapeutic agent comprises a thrombogenic orpolymerisable molecule (intended to clog the aneurysms) or proteins(elastin, fibronectin, or fibrinogen, for example), or cell growthfactors (intended to reinforce or make the fundus thicker and stronger).

In a specific embodiment, the therapeutic agent is a compound able toconvert SMCs of contractile phenotype into SMCs of synthetic phenotype.In a preferred embodiment, this compound comprises an antibody directedagainst α7β1 integrin coupled to fibronectin or fragments of fibronectin(specific fibronectin peptides). In an other embodiment, the anti-α7β1integrin antibody is coupled to fibronectin or fragments of fibronectin(specific fibronectin peptides) and Growth Factors. In contrast tocerebral aneurysm formation, neo-intima formation that occurs invascular disease such as re-stenosis after angioplasty, will be treatedor prevented by using a compound able to convert SMCs of syntheticphenotype into SMCs of contractile phenotype. In this embodiment, thetherapeutic agent is composed of antibody directed against α5β1 integrincoupled to laminin, and especially laminin-1 (FIG. 2).

E. Assembly of the Compounds

The compounds of the invention may be assembled in any suitable manner.In specific embodiments, however, Dextran is the molecule used as theintravascular targeting molecule. For nuclear imagingTechnetium-99m-Dextran is prepared according to method known in the art(Line et al., 2000) and then coupled to the cell targeting molecule(antibody or peptide). 99mTc-Dextran is restricted to the blood pool andis broken down by the liver and cleared through the kidneys. Theseproperties allow low background activity in the extra vascular space andrapid clearance from the intra vascular space. Radiolabeling through99mTc-Dextran will avoid the diffusion of the compound to the extravascular compartment. 99mTc-Dextran coupled to the cell targetingmolecule will bind specifically SMCs of the luminal surface of theaneurysmal lesion and will not bind SMCs in the medial layer of normalartery. For molecular MRI, gadolinium-DPTA-Dextran will be preparedaccording to method known in the art (Sirlin C B et al., 2004) andcoupled to the cell targeting molecule.

VII. Detection of the Compounds

Detection of a compound accumulated at the aneurysm site/or in theaneurysm vicinity may be performed according to techniques known in theart and which may vary depending on the characteristic of the labelmoiety of the conjugate. For example, when the conjugate comprises agamma-ray emitting radionuclide, the aneurysm may be detected byscintigraphy using a gamma-camera or by single photon emission computedtomography (SPECT). When the conjugate comprises a positron emittingradionuclide, the aneurysm may be detected by positron emissiontomography (PET). Combined positron emission tomography (PET) andcomputerized tomography (CT) or magnetic resonance (MR) can be used(PET/CT or PET/MR). This combined technique allows accurate detectionand localization for aneurysmal lesions. When the conjugate comprises aMRI agent contrast, the aneurysm may be detected by MRI. When theconjugate comprises a positron emitting radionuclide, the aneurysm maybe detected by positron emission tomography (PET). When the conjugate isa near infrared fluorophore (near infrared fluorescent dye), theaneurysm may be detected by near-infrared imager. When the conjugate isa microbubble contrast agent, the aneurysm may be detected by ultrasoundimagery.

Thus, different molecular imaging techniques could be used depending onthe nature of the labeling agent such as immunoscintigraphy and SPECTusing antibody radiolabeled with 99mTc-dextran or MRI using antibodyconjugated to Gadolinium-DTPA-dextran. Ultrasound may also be employed.Other techniques could use a positron emitting radionuclide and theaneurysm will be detected by positron emission tomography (PET).

VIII. Methods of Using the Compounds

In general embodiments of the invention, the compounds are employed todetect an aneurysm, and in further embodiments the compounds are used totreat an aneurysm. In certain cases the same compound is employed todetect the aneurysm as to treat the aneurysm, although in other cases adifferent compound is employed to detect the aneurysm as to treat theaneurysm. The methods may be employed in an individual suspected ofhaving an aneurysm (symptomatic aneurysm, for example), an individual atrisk for having an aneurysm (such as one with head trauma, high bloodpressure, cigarette smoking, or having certain disease states known tobe associated with an increased prevalence of aneurysm such as, but notlimited to familial intracranial aneurysm, autosomal dominant polycystickidney disease, fibrous dysplasia or coarctation of the aorta, forexample), an individual that has a history of aneurysms, or anindividual known to harbor aneurysm(s) (multiple aneurysms, forexample).

A. Diagnosis

The present invention utilizes particular compounds to detect one ormore aneurysms in an individual. In certain cases, the compound isdelivered to the individual, the compound localizes to the aneurysmsite, and the brain or a part thereof is monitored for detection of thecompound. When the compound is detected at the localized site, theindividual may be given therapy for the aneurysm, using a compound ofthe present invention having a therapeutic agent, endovascular coilembolization or surgical clip occlusion.

The detection of a signal at the major brain artery site and moreparticularly, at or near the apex of arterial forks may be indicative ofa cerebral aneurysm. A positive detection of a signal may be followed bya digital subtraction angiography in patients in need thereof. Follow-upmay be performed using the same method of diagnosis to ensure thedisappearance or reduction of any intracranial labeling after treatment.

Currently, ruptured aneurysms are diagnosed with the aid of computerizedtomography scanning followed by a 4-axle digital subtractionangiography.

Unruptured aneurysms are mainly found by serendipity during evaluationof neurological non-aneurysmal disease (incidental aneurysm), part of amultiple aneurysm constellation (multiple aneurysms), or those that aresymptomatic (symptomatic aneurysm).

Both computerized tomography scanning and magnetic resonance angiographyare poorer methods than digital subtraction angiography for detection ofaneurysms smaller than 5 mm. Such invasive method cannot be used formass detection.

Methods and compositions of the present invention that cannon-invasively detect and treat ruptured as well as unruptured aneurysmsis a more desirable embodiment and allows mass detection and preventivetreatment.

B. Therapy

In particular aspects, the present invention employs a therapeutic agenton the compound, wherein the compound targets subendothelial componentsof an aneurysmal wall. In specific embodiments, the aneurysm issuspected of being in the individual, although in other cases theaneurysm has already been detected, including by a compound of thepresent invention, for example. The therapy may be delivered to theindividual once or multiple times.

An exemplary embodiment of a therapeutic molecule may include forexample, but not limited to a compound for inducing thrombosis, acompound for promoting aneurismal wall thickening and/or a compound forpromoting cell growth. The therapeutic molecule may, more particularlybe selected from the group consisting of a thrombogenic molecule, apolymerisable molecule (intended to clog the aneurysms), a protein(e.g., elastin, fibronectin, or fibrinogen etc), and a cell growthfactor (intended to reinforce or make the fundus thicker and stronger).Another exemplary embodiment of a therapeutic molecule may include forexample a protease inhibitor, such as an elastase inhibitor or a matrixmetallo-proteinase inhibitor. Elastase inhibitors may include, withoutlimitation, alpha-1 antitrypsin, alpha-2 macroglobulin which are themain elastase inhibitors in the serum. Elafin is also a potent inhibitorof elastase and proteinase 3 which is encompassed herewith. Matrixmetallo-proteinase inhibitors may include, for example and withoutlimitation tissue inhibitors of metallo-proteinase (TIMPs) or syntheticinhibitors known in the art, such as tetracyclines and tetracyclinederivatives such as doxycycline.

IX. Combination Therapy

In order to increase the effectiveness of a compound of the invention,it may be desirable to combine these compositions with other therapieseffective in the treatment of aneurysms. More generally, these othercompositions would be provided in a combined amount effective to treatthe aneurysm. This process may involve treating the individual with thecompound of the invention and the additional therapy (drugs and/orsurgery and/or endovascular coiling) at the same time. In cases whereinthe additional therapy is a compound, as opposed to surgery and/orendovascular coiling, this may be achieved by providing the individualwith a single composition or pharmacological formulation that includesboth agents, or by delivering to the individual with the two distinctcompositions or formulations, at the same time, wherein one compositionincludes the therapy of the invention and the other includes the secondagent(s).

In the context of the present invention, it is contemplated that thecomposition of the invention could be used in conjunction with surgeryand/or endovascular coiling or drug(s). Alternatively, delivery of thecompound of the present invention may precede or follow the other agenttreatment by intervals ranging from minutes to weeks. In embodimentswhere the other agent (or surgery and/or endovascular coiling) and thecompound of the present invention are delivered separately to theindividual, one would generally ensure that a significant period of timedid not expire between the time of each delivery, such that the otheragent and compound of the invention would still be able to exert anadvantageously combined effect on the cell. In such instances, it iscontemplated that one may contact the cell with both modalities withinabout 12-24 h of each other and, more preferably, within about 6-12 h ofeach other. In some situations, it may be desirable to extend the timeperiod for treatment significantly, however, where several d (2, 3, 4,5, 6 or 7) to several wk (1, 2, 3, 4, 5, 6, 7 or 8) lapse between therespective administrations.

In some cases, the treatments are repeated as necessary.

X. Antibodies

In certain aspects of the invention, one or more antibodies are employedin the methods and compositions of the invention. These antibodies maybe used in various diagnostic or therapeutic applications, describedherein below. As used herein the term “antibody” means a polyclonalantibody, a monoclonal antibody, a chimeric antibody, a humanizedantibody, a deimmunized antibody, an antigen-binding fragment, an Fabfragment; an F(ab′)2 fragment, and Fv fragment, or a synthetic moleculecomprising an antigen-binding fragment.

In some cases, the term “antibody” is intended to refer broadly to anyimmunologic binding agent such as IgG, IgM, IgA, IgD and IgE. In certaincases, IgG and/or IgM may be utilized, because they are the most commonantibodies in the physiological situation and because they are mosteasily made in a laboratory setting.

The term “antibody” is used to refer to any antibody-like molecule thathas an antigen binding region, and includes antibody fragments such asFab′, Fab, F(ab′)2, single domain antibodies (DABs), Fv, scFv (singlechain Fv), and the like. The techniques for preparing and using variousantibody-based constructs and fragments are well known in the art. Meansfor preparing and characterizing antibodies are also well known in theart (See, e.g., Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory, 1988; incorporated herein by reference).

“Mini-antibodies” or “minibodies” are also contemplated for use with thepresent invention. Minibodies are sFv polypeptide chains which includeoligomerization domains at their C-termini, separated from the sFv by ahinge region. Pack et al. (1992) Biochem 31:1579-1584. Theoligomerization domain comprises self-associating .alpha.-helices, e.g.,leucine zippers, that can be further stabilized by additional disulfidebonds. The oligomerization domain is designed to be compatible withvectorial folding across a membrane, a process thought to facilitate invivo folding of the polypeptide into a functional binding protein.Generally, minibodies are produced using recombinant methods well knownin the art. See, e.g., Pack et al. (1992) Biochem 31:1579-1584; Cumberet al. (1992) J Immunology 149B:120-126.

Antibody-like binding peptidomimetics are also contemplated in thepresent invention. Liu et al. Cell Mol Biol (Noisy-le-grand). 2003March; 49(2):209-16 describe “antibody like binding peptidomimetics”(ABiPs), which are peptides that act as pared-down antibodies and havecertain advantages of longer serum half-life as well as less cumbersomesynthesis methods.

Monoclonal antibodies (MAbs) are recognized to have certain advantages,e.g., reproducibility and large-scale production, and their use isgenerally preferred. The invention thus provides monoclonal antibodiesof the human, murine, monkey, rat, hamster, rabbit and even chickenorigin. Due to the ease of preparation and ready availability ofreagents, murine monoclonal antibodies will often be preferred.

However, “humanized” antibodies are also contemplated, as are chimericantibodies from mouse, rat, or other species, bearing human constantand/or variable region domains, bispecific antibodies, recombinant andengineered antibodies and fragments thereof. As used herein, the term“humanized” immunoglobulin refers to an immunoglobulin comprising ahuman framework region and one or more CDR's from a non-human (usually amouse or rat) immunoglobulin. The non-human immunoglobulin providing theCDR's is called the “donor” and the human immunoglobulin providing theframework is called the “acceptor”. A “humanized antibody” is anantibody comprising a humanized light chain and a humanized heavy chainimmunoglobulin.

A. Methods for Generating Monoclonal Antibodies

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Briefly, a polyclonal antibody is prepared by immunizing an animal witha LEE or CEE composition in accordance with the present invention andcollecting antisera from that immunized animal.

A wide range of animal species can be used for the production ofantisera. Typically the animal used for production of antisera is arabbit, a mouse, a rat, a hamster, a guinea pig or a goat. The choice ofanimal may be decided upon the ease of manipulation, costs or thedesired amount of sera, as would be known to one of skill in the art.Antibodies of the invention can also be produced transgenically throughthe generation of a mammal or plant that is transgenic for theimmunoglobulin heavy and light chain sequences of interest andproduction of the antibody in a recoverable form therefrom. Inconnection with the transgenic production in mammals, antibodies can beproduced in, and recovered from, the milk of goats, cows, or othermammals. See, e.g., U.S. Pat. Nos. 5,827,690, 5,756,687, 5,750,172, and5,741,957.

As is also well known in the art, the immunogenicity of a particularimmunogen composition can be enhanced by the use of non-specificstimulators of the immune response, known as adjuvants. Suitableadjuvants include all acceptable immunostimulatory compounds, such ascytokines, chemokines, cofactors, toxins, plasmodia, syntheticcompositions or LEEs or CEEs encoding such adjuvants.

Adjuvants that may be used include IL-1, IL-2, IL-4, IL-7, IL-12,γ-interferon, GMCSP, BCG, aluminum hydroxide, MDP compounds, such asthur-MDP and nor-MDP, CGP (MTP-PE), lipid A, and monophosphoryl lipid A(MPL). RIBI, which contains three components extracted from bacteria,MPL, trehalose dimycolate (TDM) and cell wall skeleton (CWS) in a 2%squalene/Tween 80 emulsion is also contemplated. MHC antigens may evenbe used. Exemplary, often preferred adjuvants include complete Freund'sadjuvant (a non-specific stimulator of the immune response containingkilled Mycobacterium tuberculosis), incomplete Freund's adjuvants andaluminum hydroxide adjuvant.

In addition to adjuvants, it may be desirable to coadminister biologicresponse modifiers (BRM), which have been shown to upregulate T cellimmunity or downregulate suppressor cell activity. Such BRMs include,but are not limited to, Cimetidine (CIM; 1200 mg/d) (Smith/Kline, PA);low-dose Cyclophosphamide (CYP; 300 mg/m2) (Johnson/Mead, NJ), cytokinessuch as γ-interferon, IL-2, or IL-12 or genes encoding proteins involvedin immune helper functions, such as B-7.

The amount of immunogen composition used in the production of polyclonalantibodies varies upon the nature of the immunogen as well as the animalused for immunization. A variety of routes can be used to administer theimmunogen including but not limited to subcutaneous, intramuscular,intradermal, intraepidermal, intravenous and intraperitoneal. Theproduction of polyclonal antibodies may be monitored by sampling bloodof the immunized animal at various points following immunization.

A second, booster dose (e.g., provided in an injection), may also begiven. The process of boosting and titering is repeated until a suitabletiter is achieved. When a desired level of immunogenicity is obtained,the immunized animal can be bled and the serum isolated and stored,and/or the animal can be used to generate MAbs.

For production of rabbit polyclonal antibodies, the animal can be bledthrough an ear vein or alternatively by cardiac puncture. The removedblood is allowed to coagulate and then centrifuged to separate serumcomponents from whole cells and blood clots. The serum may be used as isfor various applications or else the desired antibody fraction may bepurified by well-known methods, such as affinity chromatography usinganother antibody, a peptide bound to a solid matrix, or by using, e.g.,protein A or protein G chromatography.

MAbs may be readily prepared through use of well-known techniques, suchas those exemplified in U.S. Pat. No. 4,196,265, incorporated herein byreference. Typically, this technique involves immunizing a suitableanimal with a selected immunogen composition, e.g., a purified orpartially purified protein, polypeptide, peptide or domain, be it awild-type or mutant composition. The immunizing composition isadministered in a manner effective to stimulate antibody producingcells.

The methods for generating monoclonal antibodies (MAbs) generally beginalong the same lines as those for preparing polyclonal antibodies.Rodents such as mice and rats are preferred animals, however, the use ofrabbit, sheep or frog cells is also possible. The use of rats mayprovide certain advantages (Goding, 1986, pp. 60 61), but mice arepreferred, with the BALB/c mouse being most preferred as this is mostroutinely used and generally gives a higher percentage of stablefusions.

The animals are injected with antigen, generally as described above. Theantigen may be mixed with adjuvant, such as Freund's complete orincomplete adjuvant. Booster administrations with the same antigen orDNA encoding the antigen would occur at approximately two-weekintervals.

Following immunization, somatic cells with the potential for producingantibodies, specifically B lymphocytes (B cells), are selected for usein the MAb generating protocol. These cells may be obtained frombiopsied spleens, tonsils or lymph nodes, or from a peripheral bloodsample. Spleen cells and peripheral blood cells are preferred, theformer because they are a rich source of antibody-producing cells thatare in the dividing plasmablast stage, and the latter because peripheralblood is easily accessible.

Often, a panel of animals will have been immunized and the spleen of ananimal with the highest antibody titer will be removed and the spleenlymphocytes obtained by homogenizing the spleen with a syringe.Typically, a spleen from an immunized mouse contains approximately 5×10⁷to 2×10⁸ lymphocytes.

The antibody producing B lymphocytes from the immunized animal are thenfused with cells of an immortal myeloma cell, generally one of the samespecies as the animal that was immunized. Myeloma cell lines suited foruse in hybridoma producing fusion procedures preferably are non antibodyproducing, have high fusion efficiency, and enzyme deficiencies thatrender then incapable of growing in certain selective media whichsupport the growth of only the desired fused cells (hybridomas).

Any one of a number of myeloma cells may be used, as are known to thoseof skill in the art (Goding, pp. 65 66, 1986; Campbell, pp. 75 83,1984). cites). For example, where the immunized animal is a mouse, onemay use P3 X63/Ag8, X63 Ag8.653, NS1/1.Ag 4 1, Sp210 Ag14, FO, NSO/U,MPC 11, MPC11 X45 GTG 1.7 and S194/5XX0 Bul; for rats, one may useR210.RCY3, Y3 Ag 1.2.3, IR983F and 4B210; and U 266, GM1500 GRG2, LICRLON HMy2 and UC729 6 are all useful in connection with human cellfusions. See Yoo et al., J Immunol Methods. 2002 Mar. 1; 261(1-2):1-20,for a discussion of myeloma expression systems.

One preferred murine myeloma cell is the NS-1 myeloma cell line (alsotermed P3-NS-1-Ag4-1), which is readily available from the NIGMS HumanGenetic Mutant Cell Repository by requesting cell line repository numberGM3573. Another mouse myeloma cell line that may be used is the 8azaguanine resistant mouse murine myeloma SP2/0 non producer cell line.

Methods for generating hybrids of antibody producing spleen or lymphnode cells and myeloma cells usually comprise mixing somatic cells withmyeloma cells in a 2:1 proportion, though the proportion may vary fromabout 20:1 to about 1:1, respectively, in the presence of an agent oragents (chemical or electrical) that promote the fusion of cellmembranes. Fusion methods using Sendai virus have been described byKohler and Milstein (1975; 1976), and those using polyethylene glycol(PEG), such as 37% (v/v) PEG, by Gefter et al., (1977). The use ofelectrically induced fusion methods is also appropriate (Goding pp. 7174, 1986).

Fusion procedures usually produce viable hybrids at low frequencies,about 1×10⁻⁶ to 1×10⁻⁸. However, this does not pose a problem, as theviable, fused hybrids are differentiated from the parental, unfusedcells (particularly the unfused myeloma cells that would normallycontinue to divide indefinitely) by culturing in a selective medium. Theselective medium is generally one that contains an agent that blocks thede novo synthesis of nucleotides in the tissue culture media. Exemplaryand preferred agents are aminopterin, methotrexate, and azaserine.Aminopterin and methotrexate block de novo synthesis of both purines andpyrimidines, whereas azaserine blocks only purine synthesis. Whereaminopterin or methotrexate is used, the media is supplemented withhypoxanthine and thymidine as a source of nucleotides (HAT medium).Where azaserine is used, the media is supplemented with hypoxanthine.

The preferred selection medium is HAT. Only cells capable of operatingnucleotide salvage pathways are able to survive in HAT medium. Themyeloma cells are defective in key enzymes of the salvage pathway, e.g.,hypoxanthine phosphoribosyl transferase (HPRT), and they cannot survive.The B cells can operate this pathway, but they have a limited life spanin culture and generally die within about two weeks. Therefore, the onlycells that can survive in the selective media are those hybrids formedfrom myeloma and B cells.

This culturing provides a population of hybridomas from which specifichybridomas are selected. Typically, selection of hybridomas is performedby culturing the cells by single-clone dilution in microtiter plates,followed by testing the individual clonal supernatants (after about twoto three weeks) for the desired reactivity. The assay should besensitive, simple and rapid, such as radioimmunoassays, enzymeimmunoassays, cytotoxicity assays, plaque assays, dot immunobindingassays, and the like.

The selected hybridomas would then be serially diluted and cloned intoindividual antibody producing cell lines, which clones can then bepropagated indefinitely to provide MAbs. The cell lines may be exploitedfor MAb production in two basic ways. First, a sample of the hybridomacan be injected (often into the peritoneal cavity) into ahistocompatible animal of the type that was used to provide the somaticand myeloma cells for the original fusion (e.g., a syngeneic mouse).Optionally, the animals are primed with a hydrocarbon, especially oilssuch as pristane (tetramethylpentadecane) prior to injection. Theinjected animal develops tumors secreting the specific monoclonalantibody produced by the fused cell hybrid. The body fluids of theanimal, such as serum or ascites fluid, can then be tapped to provideMAbs in high concentration. Second, the individual cell lines could becultured in vitro, where the MAbs are naturally secreted into theculture medium from which they can be readily obtained in highconcentrations.

Further, expression of antibodies of the invention (or other moietiestherefrom) from production cell lines can be enhanced using a number ofknown techniques. For example, the glutamine sythetase and DHFR geneexpression systems are common approaches for enhancing expression undercertain conditions. High expressing cell clones can be identified usingconventional techniques, such as limited dilution cloning and Microdroptechnology. The GS system is discussed in whole or part in connectionwith European Patent Nos. 0 216 846, 0 256 055, and 0 323 997 andEuropean Patent Application No. 89303964.4.

MAbs produced by either means may be further purified, if desired, usingfiltration, centrifugation and various chromatographic methods such asHPLC or affinity chromatography. Fragments of the monoclonal antibodiesof the invention can be obtained from the monoclonal antibodies soproduced by methods which include digestion with enzymes, such as pepsinor papain, and/or by cleavage of disulfide bonds by chemical reduction.Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer.

It is also contemplated that a molecular cloning approach may be used togenerate monoclonals. In one embodiment, combinatorial immunoglobulinphagemid libraries are prepared from RNA isolated from the spleen of theimmunized animal, and phagemids expressing appropriate antibodies areselected by panning using cells expressing the antigen and controlcells. The advantages of this approach over conventional hybridomatechniques are that approximately 10 ⁴ times as many antibodies can beproduced and screened in a single round, and that new specificities aregenerated by H and L chain combination which further increases thechance of finding appropriate antibodies. In another example, LEEs orCEEs can be used to produce antigens in vitro with a cell free system.These can be used as targets for scanning single chain antibodylibraries. This would enable many different antibodies to be identifiedvery quickly without the use of animals.

Another embodiment of the invention for producing antibodies accordingto the present invention is found in U.S. Pat. No. 6,091,001, whichdescribes methods to produce a cell expressing an antibody from agenomic sequence of the cell comprising a modified immunoglobulin locususing Cre-mediated site-specific recombination is disclosed. The methodinvolves first transfecting an antibody-producing cell with ahomology-targeting vector comprising a lox site and a targeting sequencehomologous to a first DNA sequence adjacent to the region of theimmunoglobulin loci of the genomic sequence which is to be converted toa modified region, so the first lox site is inserted into the genomicsequence via site-specific homologous recombination. Then the cell istransfected with a lox-targeting vector comprising a second lox sitesuitable for Cre-mediated recombination with the integrated lox site anda modifying sequence to convert the region of the immunoglobulin loci tothe modified region. This conversion is performed by interacting the loxsites with Cre in vivo, so that the modifying sequence inserts into thegenomic sequence via Cre-mediated site-specific recombination of the loxsites.

Alternatively, monoclonal antibody fragments encompassed by the presentinvention can be synthesized using an automated peptide synthesizer, orby expression of full-length gene or of gene fragments in E. coli.

B. Antibody Conjugates

The present invention further provides antibodies against contractileSMC surface proteins, polypeptides and peptides that are linked to atleast one agent to form an antibody conjugate. In order to increase theefficacy of antibody molecules as diagnostic or therapeutic agents, itis conventional to link or covalently bind or complex at least onedesired molecule or moiety. Such a molecule or moiety may be, but is notlimited to, at least one effector or reporter molecule. Effectormolecules comprise molecules having a desired activity, e.g., cytotoxicactivity. Non-limiting examples of effector molecules which have beenattached to antibodies include toxins, anti-tumor agents, therapeuticenzymes, radio-labeled nucleotides, antiviral agents, chelating agents,cytokines, growth factors, and oligo- or poly-nucleotides. By contrast,a reporter molecule is defined as any moiety which may be detected usingan assay. Non-limiting examples of reporter molecules that have beenconjugated to antibodies include enzymes, radiolabels, radionuclides,haptens, fluorescent labels, phosphorescent molecules, chemiluminescentmolecules, chromophores, luminescent molecules, photoaffinity molecules,colored particles or ligands, such as biotin.

Any antibody of sufficient selectivity, specificity or affinity may beemployed as the basis for an antibody conjugate. Such properties may beevaluated using conventional immunological screening methodology knownto those of skill in the art. Sites for binding to biological activemolecules in the antibody molecule, in addition to the canonical antigenbinding sites, include sites that reside in the variable domain that canbind pathogens, B-cell superantigens, the T cell co-receptor CD4 and theHIV-1 envelope (Sasso et al., 1989; Shorki et al., 1991; Silvermann etal., 1995; Cleary et al., 1994; Lenert et al., 1990; Berberian et al.,1993; Kreier et al., 1991). In addition, the variable domain is involvedin antibody self-binding (Kang et al., 1988), and contains epitopes(idiotopes) recognized by anti-antibodies (Kohler et al., 1989).

Certain examples of antibody conjugates are those conjugates in whichthe antibody is linked to a detectable label. “Detectable labels” arecompounds and/or elements that can be detected due to their specificfunctional properties, and/or chemical characteristics, the use of whichallows the antibody to which they are attached to be detected, and/orfurther quantified if desired. Another such example is the formation ofa conjugate comprising an antibody linked to a cytotoxic or anticellular agent, and may be termed “immunotoxins”.

Antibody conjugates are generally preferred for use as diagnosticagents. Antibody diagnostics generally fall within two classes, thosefor use in in vitro diagnostics, such as in a variety of immunoassays,and/or those for use in vivo diagnostic protocols, generally known as“antibody directed imaging”.

Many appropriate imaging agents are known in the art, as are methods fortheir attachment to antibodies (see, for e.g., U.S. Pat. Nos. 5,021,236;4,938,948; and 4,472,509, each incorporated herein by reference). Theimaging moieties used can be paramagnetic ions; radioactive isotopes;fluorochromes; NMR-detectable substances; X-ray imaging.

In the case of paramagnetic ions, one might mention by way of exampleions such as chromium (III), manganese (II), iron (III), iron (II),cobalt (II), nickel (II), copper (II), neodymium (III), samarium (III),ytterbium (III), gadolinium (III), vanadium (II), terbium (III),dysprosium (III), holmium (III) and/or erbium (III), with gadoliniumbeing particularly preferred. Ions useful in other contexts, such asX-ray imaging, include but are not limited to lanthanum (III), gold(III), lead (II), and especially bismuth (III).

In the case of radioactive isotopes for therapeutic and/or diagnosticapplication, one might mention astatine211, 14carbon, 51chromium,36chlorine, 57cobalt, 58cobalt, copper67, 152Eu, gallium67, 3hydrogen,iodine123, iodine125, iodine131, indium111, 59iron, 32phosphorus,rhenium186, rhenium188, 75selenium, 35sulphur, technicium99m and/oryttrium90. ¹²⁵I is often being preferred for use in certain embodiments,and technicium99m and/or indium111 are also often preferred due to theirlow energy and suitability for long range detection. Radioactivelylabeled monoclonal antibodies of the present invention may be producedaccording to well-known methods in the art. For instance, monoclonalantibodies can be iodinated by contact with sodium and/or potassiumiodide and a chemical oxidizing agent such as sodium hypochlorite, or anenzymatic oxidizing agent, such as lactoperoxidase. Monoclonalantibodies according to the invention may be labeled with technetium99mby ligand exchange process, for example, by reducing pertechnate withstannous solution, chelating the reduced technetium onto a Sephadexcolumn and applying the antibody to this column. Alternatively, directlabeling techniques may be used, e.g., by incubating pertechnate, areducing agent such as SNCl2, a buffer solution such as sodium-potassiumphthalate solution, and the antibody. Intermediary functional groupswhich are often used to bind radioisotopes which exist as metallic ionsto antibody are diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetracetic acid (EDTA).

Among the fluorescent labels contemplated for use as conjugates includeAlexa 350, Alexa 430, AMCA, BODIPY 630/650, BODIPY 650/665, BODIPY-FL,BODIPY-R6G, BODIPY-TMR, BODIPY-TRX, Cascade Blue, Cy3, Cy5,6-FAM,Fluorescein Isothiocyanate, HEX, 6-JOE, Oregon Green 488, Oregon Green500, Oregon Green 514, Pacific Blue, REG, Rhodamine Green, RhodamineRed, Renographin, ROX, TAMRA, TET, Tetramethylrhodamine, and/or TexasRed.

Another type of antibody conjugates contemplated in the presentinvention are those intended primarily for use in vitro, where theantibody is linked to a secondary binding ligand and/or to an enzyme (anenzyme tag) that will generate a colored product upon contact with achromogenic substrate. Examples of suitable enzymes include urease,alkaline phosphatase, (horseradish) hydrogen peroxidase or glucoseoxidase. Preferred secondary binding ligands are biotin and/or avidinand streptavidin compounds. The use of such labels is well known tothose of skill in the art and are described, for example, in U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149and 4,366,241; each incorporated herein by reference.

Yet another known method of site-specific attachment of molecules toantibodies comprises the reaction of antibodies with hapten-basedaffinity labels. Essentially, hapten-based affinity labels react withamino acids in the antigen binding site, thereby destroying this siteand blocking specific antigen reaction. However, this may not beadvantageous since it results in loss of antigen binding by the antibodyconjugate.

Molecules containing azido groups may also be used to form covalentbonds to proteins through reactive nitrene intermediates that aregenerated by low intensity ultraviolet light (Potter & Haley, 1983). Inparticular, 2- and 8-azido analogues of purine nucleotides have beenused as site-directed photoprobes to identify nucleotide bindingproteins in crude cell extracts (Owens & Haley, 1987; Atherton et al.,1985). The 2- and 8-azido nucleotides have also been used to mapnucleotide binding domains of purified proteins (Khatoon et al., 1989;King et al., 1989; and Dholakia et al., 1989) and may be used asantibody binding agents.

Several methods are known in the art for the attachment or conjugationof an antibody to its conjugate moiety. Some attachment methods involvethe use of a metal chelate complex employing, for example, an organicchelating agent such a diethylenetriaminepentaacetic acid anhydride(DTPA); ethylenetriaminetetraacetic acid; N-chloro-p-toluenesulfonamide;and/or tetrachloro-3α-6α-diphenylglycouril-3 attached to the antibody(U.S. Pat. Nos. 4,472,509 and 4,938,948, each incorporated herein byreference). Monoclonal antibodies may also be reacted with an enzyme inthe presence of a coupling agent such as glutaraldehyde or periodate.Conjugates with fluorescein markers are prepared in the presence ofthese coupling agents or by reaction with an isothiocyanate. In U.S.Pat. No. 4,938,948, imaging of breast tumors is achieved usingmonoclonal antibodies and the detectable imaging moieties are bound tothe antibody using linkers such as methyl-p-hydroxybenzimidate orN-succinimidyl-3-(4-hydroxyphenyl)propionate.

In other embodiments, derivatization of immunoglobulins by selectivelyintroducing sulfhydryl groups in the Fc region of an immunoglobulin,using reaction conditions that do not alter the antibody combining siteare contemplated. Antibody conjugates produced according to thismethodology are disclosed to exhibit improved longevity, specificity andsensitivity (U.S. Pat. No. 5,196,066, incorporated herein by reference).Site-specific attachment of effector or reporter molecules, wherein thereporter or effector molecule is conjugated to a carbohydrate residue inthe Fc region have also been disclosed in the literature (O'Shannessy etal., 1987). This approach has been reported to produce diagnosticallyand therapeutically promising antibodies which are currently in clinicalevaluation.

In another embodiment of the invention, the antibodies are linked tosemiconductor nanocrystals such as those described in U.S. Pat. Nos.6,048,616; 5,990,479; 5,690,807; 5,505,928; 5,262,357 (all of which areincorporated herein in their entireties); as well as PCT Publication No.99/26299 (published May 27, 1999). In particular, exemplary materialsfor use as semiconductor nanocrystals in the biological and chemicalassays of the present invention include, but are not limited to thosedescribed above, including group II-VI, III-V and group IVsemiconductors such as ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, MgS, MgSe,MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, GaN, GaP, GaAs,GaSb, InP, InAs, InSb, AlS, AlP, AlSb, PbS, PbSe, Ge and Si and ternaryand quaternary mixtures thereof. Methods for linking semiconductornanocrystals to antibodies are described in U.S. Pat. Nos. 6,630,307 and6,274,323.

XI. Pharmaceutical Preparations and Delivery

Pharmaceutical compositions of the present invention comprise aneffective amount of one or more compositions of the invention oradditional agent dissolved or dispersed in a pharmaceutically acceptablecarrier. The phrases “pharmaceutical or pharmacologically acceptable”refers to molecular entities and compositions that do not produce anadverse, allergic or other untoward reaction when administered to ananimal, such as, for example, a human, as appropriate. The preparationof an pharmaceutical composition that contains at least one compositionof the invention or additional active ingredient will be known to thoseof skill in the art in light of the present disclosure, as exemplifiedby Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company,1990, incorporated herein by reference. Moreover, for animal (e.g.,human) administration, it will be understood that preparations shouldmeet sterility, pyrogenicity, general safety and purity standards asrequired by FDA Office of Biological Standards.

As used herein, “pharmaceutically acceptable carrier” includes any andall solvents, dispersion media, coatings, surfactants, antioxidants,preservatives (e.g., antibacterial agents, antifungal agents), isotonicagents, absorption delaying agents, salts, preservatives, drugs, drugstabilizers, gels, binders, excipients, disintegration agents,lubricants, sweetening agents, flavoring agents, dyes, such likematerials and combinations thereof, as would be known to one of ordinaryskill in the art (see, for example, Remington's Pharmaceutical Sciences,18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated hereinby reference). Except insofar as any conventional carrier isincompatible with the active ingredient, its use in the pharmaceuticalcompositions is contemplated.

The composition of the invention may comprise different types ofcarriers depending on whether it is to be administered in solid, liquidor aerosol form, and whether it need to be sterile for such routes ofadministration as injection. The present invention can be administeredintravenously, intradermally, transdermally, intrathecally,intraarterially, intraperitoneally, intranasally, intravaginally,intrarectally, topically, intramuscularly, subcutaneously, mucosally,orally, topically, locally, inhalation (e.g., aerosol inhalation),injection, infusion, continuous infusion, localized perfusion bathingtarget cells directly, via a catheter, via a lavage, in cremes, in lipidcompositions (e.g., liposomes), or by other method or any combination ofthe forgoing as would be known to one of ordinary skill in the art (see,for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack PrintingCompany, 1990, incorporated herein by reference).

The present invention is generally intravascularly (intra-arterially orintravenously) administered, or may be delivered in situ via an arterialcatheter. The administration can be injection, infusion, or continuousinfusion.

In some embodiments of the invention, systemic injection is employed fordelivery, whereas in other embodiments of the invention there is localor in situ administration using a catheter (same technique as coiling,for example) in order to increase the local concentration of theantibody while decreasing the blood concentration in the systemicbloodstream/circulation.

In an other embodiment, a coil coated with the therapeutic compound isbrought to the aneurysm via an arterial catheter using a standardizedendovascular coiling technique.

This coil behaves as a drug delivery system and therefore realizes an insitu continuous infusion of the composition.

Once released within the aneurysm, the coated coil deploys blocking ordecreasing the blood flow into the aneurysm. These conditions allow thetopical delivery of the therapeutic agent and promote its assimilationwithin the aneurysmal wall.

This dual mechanism combines a short-term effect of clogging theaneurysmal lumen and the long-term effect of repairing the aneurysmalwall.

Both combined actions are aimed to preclude a new hemorrhage when are-permeabilization occurs after coiling. The simultaneous delivery ofthe therapeutic agent to the aneurysm will repair the aneurysmal walland prevent any hemorrhagic recurrence.

The composition of the invention may be formulated into a composition ina free base, neutral or salt form. Pharmaceutically acceptable salts,include the acid addition salts, e.g., those formed with the free aminogroups of a proteinaceous composition, or which are formed withinorganic acids such as for example, hydrochloric or phosphoric acids,or such organic acids as acetic, oxalic, tartaric or mandelic acid.Salts formed with the free carboxyl groups can also be derived frominorganic bases such as for example, sodium, potassium, ammonium,calcium or ferric hydroxides; or such organic bases as isopropylamine,trimethylamine, histidine or procaine. Upon formulation, solutions willbe administered in a manner compatible with the dosage formulation andin such amount as is therapeutically effective. The formulations areeasily administered in a variety of dosage forms such as formulated forparenteral administrations such as injectable solutions, or aerosols fordelivery to the lungs, or formulated for alimentary administrations suchas drug release capsules and the like.

Further in accordance with the present invention, the composition of thepresent invention suitable for administration is provided in apharmaceutically acceptable carrier with or without an inert diluent.The carrier should be assimilable and includes liquid, semi-solid, i.e.,pastes, or solid carriers. Except insofar as any conventional media,agent, diluent or carrier is detrimental to the recipient or to thetherapeutic effectiveness of a the composition contained therein, itsuse in administrable composition for use in practicing the methods ofthe present invention is appropriate. Examples of carriers or diluentsinclude fats, oils, water, saline solutions, lipids, liposomes, resins,binders, fillers and the like, or combinations thereof. The compositionmay also comprise various antioxidants to retard oxidation of one ormore component. Additionally, the prevention of the action ofmicroorganisms can be brought about by preservatives such as variousantibacterial and antifungal agents, including but not limited toparabens (e.g., methylparabens, propylparabens), chlorobutanol, phenol,sorbic acid, thimerosal or combinations thereof.

In accordance with the present invention, the composition is combinedwith the carrier in any convenient and practical manner, i.e., bysolution, suspension, emulsification, admixture, encapsulation,absorption and the like. Such procedures are routine for those skilledin the art.

In a specific embodiment of the present invention, the composition iscombined or mixed thoroughly with a semi-solid or solid carrier. Themixing can be carried out in any convenient manner such as grinding.Stabilizing agents can be also added in the mixing process in order toprotect the composition from loss of therapeutic activity, i.e.,denaturation in the stomach. Examples of stabilizers for use in an thecomposition include buffers, amino acids such as glycine and lysine,carbohydrates such as dextrose, mannose, galactose, fructose, lactose,sucrose, maltose, sorbitol, mannitol, etc.

In further embodiments, the present invention may concern the use of apharmaceutical lipid vehicle compositions that include composition ofthe invention, one or more lipids, and an aqueous solvent. As usedherein, the term “lipid” will be defined to include any of a broad rangeof substances that is characteristically insoluble in water andextractable with an organic solvent. This broad class of compounds arewell known to those of skill in the art, and as the term “lipid” is usedherein, it is not limited to any particular structure. Examples includecompounds which contain long-chain aliphatic hydrocarbons and theirderivatives. A lipid may be naturally occurring or synthetic (i.e.,designed or produced by man). However, a lipid is usually a biologicalsubstance. Biological lipids are well known in the art, and include forexample, neutral fats, phospholipids, phosphoglycerides, steroids,terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides,lipids with ether and ester-linked fatty acids and polymerizable lipids,and combinations thereof. Of course, compounds other than thosespecifically described herein that are understood by one of skill in theart as lipids are also encompassed by the compositions and methods ofthe present invention.

One of ordinary skill in the art would be familiar with the range oftechniques that can be employed for dispersing a composition in a lipidvehicle. For example, the composition may be dispersed in a solutioncontaining a lipid, dissolved with a lipid, emulsified with a lipid,mixed with a lipid, combined with a lipid, covalently bonded to a lipid,contained as a suspension in a lipid, contained or complexed with amicelle or liposome, or otherwise associated with a lipid or lipidstructure by any means known to those of ordinary skill in the art. Thedispersion may or may not result in the formation of liposomes.

The actual dosage amount of a composition of the present inventionadministered to an animal patient can be determined by physical andphysiological factors such as body weight, severity of condition, thetype of disease being treated, previous or concurrent therapeuticinterventions, idiopathy of the patient and on the route ofadministration. Depending upon the dosage and the route ofadministration, the number of administrations of a preferred dosageand/or an effective amount may vary according to the response of thesubject. The practitioner responsible for administration will, in anyevent, determine the concentration of active ingredient(s) in acomposition and appropriate dose(s) for the individual subject.

In certain embodiments, pharmaceutical compositions may comprise, forexample, at least about 0.1% of an active compound. In otherembodiments, the an active compound may comprise between about 2% toabout 75% of the weight of the unit, or between about 25% to about 60%,for example, and any range derivable therein. Naturally, the amount ofactive compound(s) in each therapeutically useful composition may beprepared is such a way that a suitable dosage will be obtained in anygiven unit dose of the compound. Factors such as solubility,bioavailability, biological half-life, route of administration, productshelf life, as well as other pharmacological considerations will becontemplated by one skilled in the art of preparing such pharmaceuticalformulations, and as such, a variety of dosages and treatment regimensmay be desirable.

In other non-limiting examples, a dose may also comprise from about 1microgram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milligram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 5 mg/kg/body weight to about100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

A. Alimentary Compositions and Formulations

In preferred embodiments of the present invention, the composition ofthe invention are formulated to be administered via an alimentary route.Alimentary routes include all possible routes of administration in whichthe composition is in direct contact with the alimentary tract.Specifically, the pharmaceutical compositions disclosed herein may beadministered orally, buccally, rectally, or sublingually. As such, thesecompositions may be formulated with an inert diluent or with anassimilable edible carrier, or they may be enclosed in hard- orsoft-shell gelatin capsule, or they may be compressed into tablets, orthey may be incorporated directly with the food of the diet.

In certain embodiments, the active compounds may be incorporated withexcipients and used in the form of ingestible tablets, buccal tables,troches, capsules, elixirs, suspensions, syrups, wafers, and the like(Mathiowitz et al., 1997; Hwang et al., 1998; U.S. Pat. Nos. 5,641,515;5,580,579 and 5,792,451, each specifically incorporated herein byreference in its entirety). The tablets, troches, pills, capsules andthe like may also contain the following: a binder, such as, for example,gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; anexcipient, such as, for example, dicalcium phosphate, mannitol, lactose,starch, magnesium stearate, sodium saccharine, cellulose, magnesiumcarbonate or combinations thereof; a disintegrating agent, such as, forexample, corn starch, potato starch, alginic acid or combinationsthereof; a lubricant, such as, for example, magnesium stearate; asweetening agent, such as, for example, sucrose, lactose, saccharin orcombinations thereof; a flavoring agent, such as, for examplepeppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.When the dosage unit form is a capsule, it may contain, in addition tomaterials of the above type, a liquid carrier. Various other materialsmay be present as coatings or to otherwise modify the physical form ofthe dosage unit. For instance, tablets, pills, or capsules may be coatedwith shellac, sugar, or both. When the dosage form is a capsule, it maycontain, in addition to materials of the above type, carriers such as aliquid carrier. Gelatin capsules, tablets, or pills may be entericallycoated. Enteric coatings prevent denaturation of the composition in thestomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No.5,629,001. Upon reaching the small intestines, the basic pH thereindissolves the coating and permits the composition to be released andabsorbed by specialized cells, e.g., epithelial enterocytes and Peyer'spatch M cells. A syrup of elixir may contain the active compound sucroseas a sweetening agent methyl and propylparabens as preservatives, a dyeand flavoring, such as cherry or orange flavor. Of course, any materialused in preparing any dosage unit form should be pharmaceutically pureand substantially non-toxic in the amounts employed. In addition, theactive compounds may be incorporated into sustained-release preparationand formulations.

For oral administration the compositions of the present invention mayalternatively be incorporated with one or more excipients in the form ofa mouthwash, dentifrice, buccal tablet, oral spray, or sublingualorally-administered formulation. For example, a mouthwash may beprepared incorporating the active ingredient in the required amount inan appropriate solvent, such as a sodium borate solution (Dobell'sSolution). Alternatively, the active ingredient may be incorporated intoan oral solution such as one containing sodium borate, glycerin andpotassium bicarbonate, or dispersed in a dentifrice, or added in atherapeutically-effective amount to a composition that may includewater, binders, abrasives, flavoring agents, foaming agents, andhumectants. Alternatively the compositions may be fashioned into atablet or solution form that may be placed under the tongue or otherwisedissolved in the mouth.

Additional formulations which are suitable for other modes of alimentaryadministration include suppositories. Suppositories are solid dosageforms of various weights and shapes, usually medicated, for insertioninto the rectum. After insertion, suppositories soften, melt or dissolvein the cavity fluids. In general, for suppositories, traditionalcarriers may include, for example, polyalkylene glycols, triglyceridesor combinations thereof. In certain embodiments, suppositories may beformed from mixtures containing, for example, the active ingredient inthe range of about 0.5% to about 10%, and preferably about 1% to about2%.

B. Parenteral Compositions and Formulations

In further embodiments, composition of the invention may be administeredvia a parenteral route. As used herein, the term “parenteral” includesroutes that bypass the alimentary tract. Specifically, thepharmaceutical compositions disclosed herein may be administered forexample, but not limited to intravenously, intradermally,intramuscularly, intraarterially, intrathecally, subcutaneous, orintraperitoneally U.S. Pat. Nos. 6,613,308, 5,466,468, 5,543,158;5,641,515; and 5,399,363 (each specifically incorporated herein byreference in its entirety).

Solutions of the active compounds as free base or pharmacologicallyacceptable salts may be prepared in water suitably mixed with asurfactant, such as hydroxypropylcellulose. Dispersions may also beprepared in glycerol, liquid polyethylene glycols, and mixtures thereofand in oils. Under ordinary conditions of storage and use, thesepreparations contain a preservative to prevent the growth ofmicroorganisms. The pharmaceutical forms suitable for injectable useinclude sterile aqueous solutions or dispersions and sterile powders forthe extemporaneous preparation of sterile injectable solutions ordispersions (U.S. Pat. No. 5,466,468, specifically incorporated hereinby reference in its entirety). In all cases the form must be sterile andmust be fluid to the extent that easy injectability exists. It must bestable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms, such asbacteria and fungi. The carrier can be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (i.e., glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and/or vegetable oils. Proper fluidity may bemaintained, for example, by the use of a coating, such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. The prevention of the action ofmicroorganisms can be brought about by various antibacterial andantifungal agents, for example, parabens, chlorobutanol, phenol, sorbicacid, thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars or sodium chloride.Prolonged absorption of the injectable compositions can be brought aboutby the use in the compositions of agents delaying absorption, forexample, aluminum monostearate and gelatin.

For parenteral administration in an aqueous solution, for example, thesolution should be suitably buffered if necessary and the liquid diluentfirst rendered isotonic with sufficient saline or glucose. Theseparticular aqueous solutions are especially suitable for intravenous,intramuscular, subcutaneous, and intraperitoneal administration. In thisconnection, sterile aqueous media that can be employed will be known tothose of skill in the art in light of the present disclosure. Forexample, one dosage may be dissolved in isotonic NaCl solution andeither added hypodermoclysis fluid or injected at the proposed site ofinfusion, (see for example, “Remington's Pharmaceutical Sciences” 15thEdition, pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated. The person responsible for administration will, in any event,determine the appropriate dose for the individual subject. Moreover, forhuman administration, preparations should meet sterility, pyrogenicity,general safety and purity standards as required by FDA Office ofBiologics standards.

Sterile injectable solutions are prepared by incorporating the activecompounds in the required amount in the appropriate solvent with variousof the other ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the requiredother ingredients from those enumerated above. In the case of sterilepowders for the preparation of sterile injectable solutions, thepreferred methods of preparation are vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof. A powdered composition is combined with a liquidcarrier such as, e.g., water or a saline solution, with or without astabilizing agent.

C. Miscellaneous Pharmaceutical Compositions and Formulations

In other preferred embodiments of the invention, the active compound maybe formulated for administration via various miscellaneous routes, forexample, topical (i.e., transdermal) administration, mucosaladministration (intranasal, vaginal, etc.) and/or inhalation.

Pharmaceutical compositions for topical administration may include theactive compound formulated for a medicated application such as anointment, paste, cream or powder. Ointments include all oleaginous,adsorption, emulsion and water-solubly based compositions for topicalapplication, while creams and lotions are those compositions thatinclude an emulsion base only. Topically administered medications maycontain a penetration enhancer to facilitate adsorption of the activeingredients through the skin. Suitable penetration enhancers includeglycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones andluarocapram. Possible bases for compositions for topical applicationinclude polyethylene glycol, lanolin, cold cream and petrolatum as wellas any other suitable absorption, emulsion or water-soluble ointmentbase. Topical preparations may also include emulsifiers, gelling agents,and antimicrobial preservatives as necessary to preserve the activeingredient and provide for a homogenous mixture. Transdermaladministration of the present invention may also comprise the use of a“patch”. For example, the patch may supply one or more active substancesat a predetermined rate and in a continuous manner over a fixed periodof time.

In certain embodiments, the pharmaceutical compositions may be deliveredby eye drops, intranasal sprays, inhalation, and/or other aerosoldelivery vehicles. Methods for delivering compositions directly to thelungs via nasal aerosol sprays has been described e.g., in U.S. Pat.Nos. 5,756,353 and 5,804,212 (each specifically incorporated herein byreference in its entirety). Likewise, the delivery of drugs usingintranasal microparticle resins (Takenaga et al., 1998) andlysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871,specifically incorporated herein by reference in its entirety) are alsowell-known in the pharmaceutical arts. Likewise, transmucosal drugdelivery in the form of a polytetrafluoroetheylene support matrix isdescribed in U.S. Pat. No. 5,780,045 (specifically incorporated hereinby reference in its entirety).

The term aerosol refers to a colloidal system of finely divided solid ofliquid particles dispersed in a liquefied or pressurized gas propellant.The typical aerosol of the present invention for inhalation will consistof a suspension of active ingredients in liquid propellant or a mixtureof liquid propellant and a suitable solvent. Suitable propellantsinclude hydrocarbons and hydrocarbon ethers. Suitable containers willvary according to the pressure requirements of the propellant.Administration of the aerosol will vary according to subject's age,weight and the severity and response of the symptoms.

XII. Kits

Any of the compositions described herein may be comprised in a kit forthe detection and/or treatment of aneurysm. The kit may include a celltargeting molecule, a label, an intravascular targeting molecule, and/ora therapeutic agent. These components may or may not be assembled intoone composition. In a non-limiting example, there may be an additionalagent provided in the kit. The kits may thus comprise, in suitablecontainer means, a composition of the invention and, optionally, anadditional agent of the present invention. Kits of the present inventionwill generally contain, in suitable container means, a pharmaceuticallyacceptable formulation of the composition of the invention.

The component(s) of the kits may be packaged either in aqueous media orin lyophilized form. When reagents and/or components are provided as adry powder, the powder can be reconstituted by the addition of asuitable solvent. It is envisioned that the solvent may also be providedin another container means. The container means of the kits willgenerally include at least one vial, test tube, flask, bottle, syringeor other container means, into which a component may be placed, andpreferably, suitably aliquoted. Where there are more than one componentin the kit, the kit also will generally contain a second, third or otheradditional container into which the additional components may beseparately placed. However, various combinations of components may becomprised in a vial. The kits of the present invention also willtypically include a means for containing the composition of theinvention and any other reagent containers in close confinement forcommercial sale. Such containers may include injection or blow moldedplastic containers into which the desired vials are retained.

EXAMPLES

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Exemplary Experimental Designs and Methods

The present example provides exemplary materials and methods for theinvention, although one of skill in the art recognizes that they aremerely exemplary in nature and may be modified within routine standardsin the art.

1. In Situ Immunohistochemistry

Animal Preparation

Intracranial aneurysms are induced in 20 male Sprague-Dawley rats (agerange, 6 to 7 weeks) according to the method of Hashimoto et al(Hashimoto et al., 1978). Ligation of the left common carotid artery andthe posterior branches of both renal arteries will be performed underanesthesia with the use of an intraperitoneal injection of chloralhydrate (3%, 0.01 mL/g body wt). One week after the operation, 1% salinewill be substituted for drinking water. The previous literature reportedthis method of preferentially induced experimental cerebral aneurysms atthe right anterior cerebral artery (ACA)-olfactory artery (OA)bifurcations, where hemodynamic stress is assumed to increase by theligation of the opposite common carotid artery (Kojima et al., 1986). Anadditional 5 age-matched rats will served as controls.

Three months after the aneurysm induction procedure, the rats will becannulated into the ascending aorta through the left cardiac ventricleunder general anesthesia and perfused at a pressure of 80 mmHg with 4%paraformaldehyde in PBS. After the perfusion fixations, the majorarteries at the base of the brain will be carefully dissected under asurgical microscope. The specimens will be further immersed in 4%paraformaldehyde in PBS for 24 hours. The specimens will be rinsed withPBS, embedded in OCT compound (Tissue-Tek, Inc) and 7 μm-thick serialsections from ACA/OlfA bifurcation will be cut with a cryotome.

Light Microscopic Examination

Using elastica-van Gieson stain and a light microscope, we will examinethe bifurcation of the ACA and OA on both sides and examine aneurismalchanges on the nonligated side.

Definition of Aneurysmal Changes

It is established that fragmentation and disappearance of internalelastic lamina is a characteristic histological feature of aneurysmallesions. Therefore, aneurysmal changes will be defined as lesionsrepresenting the outward dilatation of the wall that are accompanied bydiscontinuity of the internal elastic lamina in more than half thelength of the dilated wall (evidenced by elastica-van Gieson staining).The lesions will be classified into two stages: (1) a stage of earlyaneurismal lesion preserving the smooth cell layer in the whole area ofthe wall and (2): saccular aneurysm lacking the smooth muscle layer evenin part of the whole area of the lesion. Moreover, to detect endothelialinjury at the apical intimal pad which is characteristic of earlyaneurismal lesion, we will performed immunohistochemical study usingantibody against eNOS (endothelial injury is evidenced by the loss ofeNOS expression).

Immunohistochemical Studies

Immunohistochemical studies will be performed on early aneurismallesions and saccular aneurysm to study α7 integrin and laminin-1expression. Our goal is to demonstrate that early aneurismal lesionsshow a lack of eNOS expression and a subendothelial staining for α7integrin and laminin-1 in contrast to control sections that show apositive expression of eNOS and a medial without subendothelial stainingfor α7 integrin and laminin-1. Sections will be fixed in ice-coldacetone (10 minutes), air-dried (30 minutes), and incubated in 5% skimmilk (30 minutes) before overnight incubation at 4° C. with primaryantibodies. The primary antibodies will be mouse anti-eNOS antibody (BDBiosciences), mouse anti-rat α7 integrin antibody (H36 provide by SJKaufman) rabbit polyclonal anti-laminin α-1 (H-300) (sc-5582, Santa CruzBiotechnology, Inc). Sections will be washed with PBS, then incubated 30minutes with fluorescein-conjugated secondary antibody (Alexa Fluor 594,488 and 647 goat anti-mouse, anti-rabbit or anti-rat immunoglobulin G;Molecular Probes). For double immunofluorescence staining (eNOS and α7,eNOs and laminin-1) the same procedure will be repeated. After washingwith PBS, the specimens will be mounted with Vectashield (VectorLaboratories).

Slides will be inspected under a fluorescent microscope combined with alaser confocal system. Image files will be digitally processed usingAdobe Photoshop (Adobe Systems).

2. In Vivo Immunodetection of Aneurysmal Lesions

Molecular Imaging techniques well suitable for intravascularapplications will be used such as immunoscintigraphy using antibodyradiolabeled with 99mTc-dextran and/or MRI using antibody conjugated toGadolinium-DTPA-dextran.

Dextran is a hydrophilic molecule that does not pass the phospholipidbilayer of endothelial membrane. Antibody conjugated to dextran will notpass the endothelium. The conjugate will bind specifically α7 integrinor laminin-1 exposed at the luminal surface of the aneurysmal wall andcan not bind α7 integrin or laminin-1 on SMCs in the medial layer ofnormal vascular wall that harbors an intact endothelium. Dextran isuseful in the invention, but any macromolecular agent with intravascularretention may be coupled to the antibody.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

PATENTS AND PATENT APPLICATIONS

U.S. Pat. No. 3,817,837

U.S. Pat. No. 3,850,752

U.S. Pat. No. 3,939,350

U.S. Pat. No. 3,996,345

U.S. Pat. No. 4,196,265

U.S. Pat. No. 4,275,149

U.S. Pat. No. 4,277,437

U.S. Pat. No. 4,366,241

U.S. Pat. No. 4,472,509

U.S. Pat. No. 4,938,948

U.S. Pat. No. 5,021,236

U.S. Pat. No. 5,741,957

U.S. Pat. No. 5,750,172

U.S. Pat. No. 5,756,687

U.S. Pat. No. 5,827,690

U.S. Pat. No. 6,091,001

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Although the present invention and its advantages have been described indetail, it should be understood that various changes, substitutions andalterations can be made herein without departing from the spirit andscope of the invention as defined by the appended claims. Moreover, thescope of the present application is not intended to be limited to theparticular embodiments of the process, machine, manufacture, compositionof matter, means, methods and steps described in the specification. Asone of ordinary skill in the art will readily appreciate from thedisclosure of the present invention, processes, machines, manufacture,compositions of matter, means, methods, or steps, presently existing orlater to be developed that perform substantially the same function orachieve substantially the same result as the corresponding embodimentsdescribed herein may be utilized according to the present invention.Accordingly, the appended claims are intended to include within theirscope such processes, machines, manufacture, compositions of matter,means, methods, or steps.

1-45. (canceled)
 46. A method of treating an aneurysm in an individual,comprising the step of delivering to the individual having or suspectedof having a cerebral aneurysm a composition comprising a) a labeledantibody that specifically binds to at least one integrin expressed oncontractile SMCs, wherein the labeled antibody localizes to the cerebralaneurysm site, b) a pharmaceutically acceptable excipient, and c) atherapeutic agent, in an amount effective to treat the aneurysm in theindividual.
 47. The method of claim 46, wherein the individual has anunruptured cerebral aneurysm.
 48. The method of claim 46, wherein theindividual has a ruptured cerebral aneurysm.
 49. The method of claim 46,wherein the therapeutic agent is selected from the group consisting of:a thrombogenic agent, a polymerisable molecule, a protein, a cell growthfactor, and a protease inhibitor.
 50. The method of claim 49, whereinthe therapeutic moiety is selected from the group consisting of:elastin, elastin degradation fragment, fibronectin, fibrinogen, and acell growth factor.
 51. The method of claim 49, wherein the therapeuticagent is a protease inhibitor.
 52. The method of claim 51, wherein theprotease inhibitor is an elastase inhibitor or a matrixmetalloproteinase inhibitor.
 53. The method of claim 46, wherein thetherapeutic agent comprises a protease inhibitor and fibronectin orfibrinogen.
 54. The method of claim 46, wherein the therapeutic moietycomprises fibronectin and a cell growth factor.
 55. The method of claim46, wherein the composition is administered to the individualintravascularly.
 56. The method of claim 46, wherein administration ofthe composition results in accumulation of the labeled antibody andtherapeutic agent at the aneurysm wall and thrombosis or aneurism wallthickening.
 57. The method of claim 46, wherein the at least oneintegrin is one or more selected from the group consisting of: α1β1,α7β1, α3β1, and α8β1.
 58. The method of claim 46, wherein thecomposition is delivered in situ via an arterial catheter.
 59. Themethod of claim 46, wherein the labeled antibody is conjugated to anintravascular targeting molecule.
 60. The method of claim 59, whereinthe intravascular targeting molecule is a polymer.
 61. The method ofclaim 60, wherein the polymer is albumin, transferrin, a globulin,pectin, gelatin, dextran, or a cellulose derivative.
 62. The method ofclaim 46, further comprising administering to the individual a drug orperforming surgery or endovascular coiling on the individual.